Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains of Pseudomonas fluorescens

Pseudomonas fluorescens CHA0 and the related strain Pf-5 are well-characterized representatives of rhizosphere bacteria that have the capacity to protect crop plants from fungal root diseases, mainly by releasing a variety of exoproducts that are toxic to plant pathogenic fungi. Here, we report that the two plant-beneficial pseudomonads also exhibit potent insecticidal activity. Anti-insect activity is linked to a novel genomic locus encoding a large protein toxin termed Fit (for P. fluorescensinsecticidal toxin) that is related to the insect toxin Mcf (Makes caterpillars floppy) of the entomopathogen Photorhabdus luminescens, a mutualist of insect-invading nematodes. When injected into the haemocoel, even low doses of P. fluorescens CHA0 or Pf-5 killed larvae of the tobacco hornworm Manduca sexta and the greater wax moth Galleria mellonella. In contrast, mutants of CHA0 or Pf-5 with deletions in the Fit toxin gene were significantly less virulent to the larvae. When expressed from an inducible promoter in a non-toxic Escherichia coli host, the Fit toxin gene was sufficient to render the bacterium toxic to both insect hosts. Our findings establish the Fit gene products of P. fluorescens CHA0 and Pf-5 as potent insect toxins that define previously unappreciated anti-insect properties of these plant-colonizing bacteria

Cross-scale analysis of the region effect on vascular plant species diversity in southern and northern European mountain ranges.

Background: The divergent glacial histories of southern and northern Europe affect present-day species diversity at coarse-grained scales in these two regions, but do these effects also penetrate to the more fine-grained scales of local communities?Methodology/Principal Findings: We carried out a cross-scale analysis to address this question for vascular plants in two mountain regions, the Alps in southern Europe and the Scandes in northern Europe, using environmentally paired vegetation plots in the two regions (n = 403 in each region) to quantify four diversity components: (i) total number of species occurring in a region (total gamma-diversity), (ii) number of species that could occur in a target plot after environmental filtering (habitat-specific gamma-diversity), (iii) pair-wise species compositional turnover between plots (plot-to-plot beta-diversity) and (iv) number of species present per plot (plot gamma-diversity). We found strong region effects on total gamma-diversity, habitat-specific gamma-diversity and plot-to-plot beta-diversity, with a greater diversity in the Alps even towards distances smaller than 50 m between plots. In contrast, there was a slightly greater plot alpha-diversity in the Scandes, but with a tendency towards contrasting region effects on high and low soil-acidity plots.Conclusions/Significance: We conclude that there are strong regional differences between coarse-grained (landscape- to regional-scale) diversity components of the flora in the Alps and the Scandes mountain ranges,but that these differences do not necessarily penetrate to the finest-grained (plot-scale) diversity component, at least not on acidic soils. Because different processes can lead to a similar pattern, we discuss the consistency of our results with Quaternary history and other divergent features between the two regions such as habitat connectivity, selection for vagility and environmental differences not accounted for in our analyses

Characterization and functional identification of a novel plant extradiol 4,5-dioxygenase involved in betalain pigment biosynthesis in Portulaca Grandiflora

RESUME Les bétalaïnes sont des pigments chromo-alcaloïdes violets et jaunes présents dans les plantes appartenant à l'ordre des Caryophyllales et dans les champignons des genres Amanita et Hygrocybe. Leur courte voie de biosynthèse est élucidée chimiquement depuis de nombreuses années, mais les enzymes impliquées dans cette biosynthèse chez les plantes ne sont toujours pas caractérisées. L'enzyme de la DOPA-dioxygénase d' Amanita muscaria a été identifiée (Girod et Zryd, 1991a), mais de nombreuses tentatives d'isolation d'un homologue chez les plantes ont échoué. Afin d'isoler les gènes spécifiques des bétalaïnes chez les plantes, nous avons construit des banques soustraites d'ADNc à partir d'ARN total de pétales immatures de Portulaca grandiflora (Pg) de génotypes jaunes et blancs, respectivement violets et blancs. Les clones couleur- spécifiques ont été détectés en premier par analyse Northem du RNA de pétales blancs et colorés. Les candidats positifs ont alors été soumis à une analyse de transcription au niveau des tiges colorées, vertes et des feuilles, afin d'établir leur expression spécifique. Deux ARNs messagers complets ont une expression corrélée avec l'accumulation des bétalaïnes dans les tissus. Le premier de ces clones, A.16, code pour une oxydase de l'acyl-Coenzyme A (ACX) putative, mais le domaine de liaison du FAD essentiel pour l'activité d'ACX est absent. Toutes nos tentatives pour démontrer sa fonction ont échoué. Le rôle de cette protéine dans la voie de synthèse des bétalaïnes reste inconnu. Le deuxième de ces clones spécifique aux bétalaïnes, L.6 (isolé par Zaiko, 2000), a été renommé DODA en raison de son homologie avec le domaine LigB (pfam02900) d'une 4,5-dioxygénase extradiol bactérienne. DODA a été identifié in silico comme une dioxygénase extradiol en raison de la conservation stricte, au niveau de sa séquence peptidique, des résidus catalytiques de LigB et de ceux liant le cofacteur fer. Une analyse de transfert Southem a montré que ce gène est unique dans Pg. L'expression transitoire de DODA par transformation biolistique dans des pétales blancs de Pg a produit des taches violettes ou jaunes dans des cellules transformées. Une analyse HPLC de ces taches a démontré leur identité avec les bétalaïnes présentes naturellement dans les pétales violets et jaunes de Pg, confirmant ainsi la complémentation par le gène Pg DODA de l'allèle récessif cc présent dans les pétales blancs de Pg. Des homologues de DODA (DOPA-dioxygénase) ont été identifiés dans de nombreuses espèces de plantes, y compris dans celles sans bétalaïne. L'alignement de ces homologues a permis l'identification d'un motif spécifique aux bétalaïnes à côté d'une histidine catalytique conservée. Ce motif [H-P-(S,A)-(N,D)-x-T-P] remplace le motif [H-N-L-R] conservé dans les plantes sans bétalaïne et le motif [H-N-L-x] présent dans tous les homologues bactériens et archaebactériens. Une modélisation tridimensionnelle préliminaire du site actif de Pg DODA et de son homologue dans la mousse Physcomitrella patens a montré l'importance de ce motif spécifique aux bétalaïnes pour l'accessibilité du substrat au site actif. L'analyse phylogénétique de DODA a confirmé l'évolution séparée de cette protéine chez les plantes à bétalaïnes par comparaison avec celle des plantes sans bétalaïne. Nous avons donc conclu que les bétalaïnes sont apparues par modification de l'affinité pour un substrat d'enzymes similaires à DODA, chez un ancêtre unique des Caryophyllales qui a perdu toute capacité de biosynthèse des anthocyanes. Finalement, Pg DODA n'a aucune similarité avec la protéine DODA d' Amanita muscaria, bien que celle-ci complémente aussi la pigmentation des pétales blancs de Pg. La biosynthèse des bétalaïnes est un exemple remarquable de convergence évolutive biochimique indépendante entre espèces de règnes différents. ABSTRACT Betalains are violet and yellow chromo-alkaloid pigments present in plants belonging to the order Caryophyllales and also in the fungal genera Amanita and Hygrocybe. Their short biosynthetic pathway is chemically well understood since many years, but enzymes involved in the plant pathway are still uncharacterized. The DOPA-dioxygenase from Amanita muscaria was identified (Girod and Zryd, 1991a), but numerous attempts to identify a plant homologue to the corresponding gene, failed. In order to isolate betalain-specific genes in plants, subtractive cDNA libraries were built with total RNA from white and yellow and respectively, violet immature petals from Portulaca grandiflora (Pg) genotypes. Colour-specific clones were first detected by Northern blot analysis using RNA from white and coloured petals. Positive candidates were submitted to further transcription analysis in coloured, green stems and leaves in order to assess their specific expression. Two full-length mRNAs showed a correlated expression with betalain accumulation in tissues. One of them, A.16, encodes a putative acyl-Coenzyme A oxidase (ACX), but missing the FAD binding domain essential for the ACX activity. Thus, all attempts to demonstrate its function failed. The role of this protein in the betalain biosynthesis pathway, if any, is still unknown. The second betalain-specific mRNA, L.6 (isolated by Zaiko, 2000) shows a homology with a LigB domain (pfam02900) from a bacterial extradiol 4,5-dioxygenase. It was then renamed DODA (DOPA-dioxygenase). DODA was identified in silico as a highly conserved extradiol dioxygenase due to the strict conservation of its peptidic sequence with LigB catalytic residues and iron-binding cofactor residues. Southern blot analysis showed that this gene is a single copy-gene in Pg. Transient expression of DODA protein through biolistic transformation of Pg white petals produced violet or yellow spots in individual cells. HPLC analysis of these spots showed an identity with betalain pigments present naturally in yellow and violet Pg petals, thus confirming the complementation of the recessive cc allele present in Pg white petals by Pg DODA gene. DODA homologues were identified in numerous plant species including those without betalain. Alignment of these homologues allowed the identification of a betalain-specific pattern beside a highly conserved catalytic histidine. This [H-P-(S,A)-(N,D)-x-T-P] pattern replaces a [H-N-L-R] pattern strictly conserved in non-betalain plants and a [H-N-L-x] pattern present in all bacterial and archaebacterial homologues. Preliminary three-dimensional modeling of the active site of Pg DODA and its Physcomitrella patens moss homologue revealed the importance of this betalain-specific pattern for the substrate accessibility to the DODA active site. DODA phylogenetic analysis confirmed the separate evolution of this protein in betalain-producing plants. We conclude that betalain pigments appeared in a unique ancestor of the Caryophyllales order in which anthocyanin biosynthetic pathway was impaired, by a modification of enzymes of the DODA family for substrate affinity. The Pg DODA protein has no sequence similarity with Amanita muscaria DODA, despite the fact that they both complement Pg white petals for their pigmentation. Betalain biosynthesis is an interesting example of independent biochemical evolutionary convergence between species from different kingdoms.

Clonal growth and demography of a hemicryptophyte alpine plant: Leontopodium alpinum Cassini

Clonally reproducing hemicryptophytic rosette plants are common in the alpine belt. However, their demography, and indirectly their growth and reproductive strategy in these harsh conditions, was rarely studied. We analysed the morphology, clonal reproduction and demography of one such species, Leontopodium alpinum, in two populations of the Swiss Alps. The species forms small colonies of 1-5 (maximum 30) sterile rosettes with a few flowering stalks. After flowering, the apical meristem dies and one or two new axillary buds grow below the previous rosette in the following year, developing into short rhizomes (<2 cm), which decay after four years. The new stalk produces sterile rosettes before flowering after two to four years, depending on climatic conditions. The apical meristem often dies during the sterile stage, and is replaced by a new axillary bud. Levkovitch matrices on two stages (sterile and flowering rosettes) showed that rosette survival and clonal reproduction maintain long-lived populations (λ = 0.96). Elasticities indicated that a change in the survival of sterile rosettes had the strongest effect on population dynamics, and this stage lasts, on average, 6.8 years at 2480 m. Altogether, L. alpinum is following Tomlinson's architectural model. This growth form appears perfectly adapted to harsh alpine conditions: the clonal ramification ensures longevity to genets and the semelparous behaviour of the rosettes allows an efficient flowering, whatever the climatic conditions. L. alpinum appears to follow a common growth model among rosette possessing hemicryptophytes in the alpine belt.

Tree cover at fine and coarse spatial grains interacts with shade tolerance to shape plant species distributions across the Alps

The role of competition for light among plants has long been recognized at local scales, but its potential importance for plant species' distribution at larger spatial scales has largely been ignored. Tree cover acts as a modulator of local abiotic conditions, notably by reducing light availability below the canopy and thus the performance of species that are not adapted to low-light conditions. However, this local effect may propagate to coarser spatial grains. Using 6,935 vegetation plots located across the European Alps, we fit Generalized Linear Models (GLM) for the distribution of 960 herbs and shrubs species to assess the effect of tree cover at both plot and landscape grain sizes (~ 10-m and 1-km, respectively). We ran four models with different combinations of variables (climate, soil and tree cover) for each species at both spatial grains. We used partial regressions to evaluate the independent effects of plot- and landscape-scale tree cover on plant communities. Finally, the effects on species' elevational range limits were assessed by simulating a removal experiment comparing the species' distribution under high and low tree cover. Accounting for tree cover improved model performance, with shade-tolerant species increasing their probability of presence at high tree cover whereas shade-intolerant species showed the opposite pattern. The tree cover effect occurred consistently at both plot and landscape spatial grains, albeit strongest at the former. Importantly, tree cover at the two grain sizes had partially independent effects on plot-scale plant communities, suggesting that the effects may be transmitted to coarser grains through meta-community dynamics. At high tree cover, shade-intolerant species exhibited elevational range contractions, especially at their upper limit, whereas shade-tolerant species showed elevational range expansions at both limits. Our findings suggest that the range shifts for herb and shrub species may be modulated by tree cover dynamics.

Plant Phototropic Growth.

Plants are photoautotrophic sessile organisms that use environmental cues to optimize multiple facets of growth and development. A classic example is phototropism - in shoots this is typically positive, leading to growth towards the light, while roots frequently show negative phototropism triggering growth away from the light. Shoot phototropism optimizes light capture of leaves in low light environments and hence increases photosynthetic productivity. Phototropins are plasma-membrane-associated UV-A/blue-light activated kinases that trigger phototropic growth. Light perception liberates their protein kinase domain from the inhibitory action of the amino-terminal photosensory portion of the photoreceptor. Following a series of still poorly understood events, phototropin activation leads to the formation of a gradient of the growth hormone auxin across the photo-stimulated stem. The greater auxin concentration on the shaded compared with the lit side of the stem enables growth reorientation towards the light. In this Minireview, we briefly summarize the signaling steps starting from photoreceptor activation until the establishment of a lateral auxin gradient, ultimately leading to phototropic growth in shoots.

Plant sex chromosomes: lost genes with little compensation.

In many animals, gene loss on Y chromosomes is compensated through altered expression of their X-chromosome homologue. Now, however, a new study in plants finds that even genes deleted from the Y show no dosage compensation.

Plant Mating Systems: Female Sterility in the Driver's Seat.

Violation of Mendel's Law of Segregation by selfish X chromosomes that favour their own transmission is known for a number of organisms. Now, a new study reveals sex-ratio distortion favouring males and explains previously puzzling sex ratios in a Mediterranean shrub.

Back to Gondwanaland: can ancient vicariance explain (some) Indian Ocean disjunct plant distributions?

Oceans, or other wide expanses of inhospitable environment, interrupt present day distributions of many plant groups. Using molecular dating techniques, generally incorporating fossil evidence, we can estimate when such distributions originated. Numerous dating analyses have recently precipitated a paradigm shift in the general explanations for the phenomenon, away from older geological causes, such as continental drift, in favour of more recent, long-distance dispersal (LDD). For example, the 'Gondwanan vicariance' scenario has been dismissed in various studies of Indian Ocean disjunct distributions. We used the gentian tribe Exaceae to reassess this scenario using molecular dating with minimum (fossil), maximum (geological), secondary (from wider analyses) and hypothesis-driven age constraints. Our results indicate that ancient vicariance cannot be ruled out as an explanation for the early origins of Exaceae across Africa, Madagascar and the Indian subcontinent unless a strong assumption is made about the maximum age of Gentianales. However, both the Gondwanan scenario and the available evidence suggest that there were also several, more recent, intercontinental dispersals during the diversification of the group.

Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses.

The epidermis on leaves protects plants from pathogen invasion and provides a waterproof barrier. It consists of a layer of cells that is surrounded by thick cell walls, which are partially impregnated by highly hydrophobic cuticular components. We show that the Arabidopsis T-DNA insertion mutants of REDUCED WALL ACETYLATION 2 (rwa2), previously identified as having reduced O-acetylation of both pectins and hemicelluloses, exhibit pleiotrophic phenotype on the leaf surface. The cuticle layer appeared diffused and was significantly thicker and underneath cell wall layer was interspersed with electron-dense deposits. A large number of trichomes were collapsed and surface permeability of the leaves was enhanced in rwa2 as compared to the wild type. A massive reprogramming of the transcriptome was observed in rwa2 as compared to the wild type, including a coordinated up-regulation of genes involved in responses to abiotic stress, particularly detoxification of reactive oxygen species and defense against microbial pathogens (e.g., lipid transfer proteins, peroxidases). In accordance, peroxidase activities were found to be elevated in rwa2 as compared to the wild type. These results indicate that cell wall acetylation is essential for maintaining the structural integrity of leaf epidermis, and that reduction of cell wall acetylation leads to global stress responses in Arabidopsis.

Plant Immune Responses: Aphids Strike Back.

To survive and complete their life cycle, herbivorous insects face the difficult challenge of coping with the arsenal of plant defences. A new study reports that aphids secrete evolutionarily conserved cytokines in their saliva to suppress host immune responses.

Phylogeny and biogeography of Primula sect. Armerina: implications for plant evolution under climate change and the uplift of the Qinghai-Tibet Plateau.

BACKGROUND: The historical orogenesis and associated climatic changes of mountain areas have been suggested to partly account for the occurrence of high levels of biodiversity and endemism. However, their effects on dispersal, differentiation and evolution of many groups of plants are still unknown. In this study, we examined the detailed diversification history of Primula sect. Armerina, and used biogeographic analysis and macro-evolutionary modeling to investigate a series of different questions concerning the evolution of the geographical and ecological distribution of the species in this section. RESULTS: We sequenced five chloroplast and one nuclear genes for species of Primula sect. Armerina. Neither chloroplast nor nuclear trees support the monophyly of the section. The major incongruences between the two trees occur among closely related species and may be explained by hybridization. Our dating analyses based on the chloroplast dataset suggest that this section began to diverge from its relatives around 3.55 million years ago, largely coinciding with the last major uplift of the Qinghai-Tibet Plateau (QTP). Biogeographic analysis supports the origin of the section in the Himalayan Mountains and dispersal from the Himalayas to Northeastern QTP, Western QTP and Hengduan Mountains. Furthermore, evolutionary models of ecological niches show that the two P. fasciculata clades have significantly different climatic niche optima and rates of niche evolution, indicating niche evolution under climatic changes and further providing evidence for explaining their biogeographic patterns. CONCLUSION: Our results support the hypothesis that geologic and climatic events play important roles in driving biological diversification of organisms in the QTP area. The Pliocene uplift of the QTP and following climatic changes most likely promoted both the inter- and intraspecific divergence of Primula sect. Armerina. This study also illustrates how niche evolution under climatic changes influences biogeographic patterns.

Evolutionary patchwork of an insecticidal toxin shared between plant-associated pseudomonads and the insect pathogens Photorhabdus and Xenorhabdus.

BACKGROUND: Root-colonizing fluorescent pseudomonads are known for their excellent abilities to protect plants against soil-borne fungal pathogens. Some of these bacteria produce an insecticidal toxin (Fit) suggesting that they may exploit insect hosts as a secondary niche. However, the ecological relevance of insect toxicity and the mechanisms driving the evolution of toxin production remain puzzling. RESULTS: Screening a large collection of plant-associated pseudomonads for insecticidal activity and presence of the Fit toxin revealed that Fit is highly indicative of insecticidal activity and predicts that Pseudomonas protegens and P. chlororaphis are exclusive Fit producers. A comparative evolutionary analysis of Fit toxin-producing Pseudomonas including the insect-pathogenic bacteria Photorhabdus and Xenorhadus, which produce the Fit related Mcf toxin, showed that fit genes are part of a dynamic genomic region with substantial presence/absence polymorphism and local variation in GC base composition. The patchy distribution and phylogenetic incongruence of fit genes indicate that the Fit cluster evolved via horizontal transfer, followed by functional integration of vertically transmitted genes, generating a unique Pseudomonas-specific insect toxin cluster. CONCLUSIONS: Our findings suggest that multiple independent evolutionary events led to formation of at least three versions of the Mcf/Fit toxin highlighting the dynamic nature of insect toxin evolution.