Full-genome sequence of the plant growth-promoting bacterium Pseudomonas protegens CHA0.

We report the complete genome sequence of the free-living bacterium Pseudomonas protegens (formerly Pseudomonas fluorescens) CHA0, a model organism used in plant-microbe interactions, biological control of phytopathogens, and bacterial genetics.

Signal transduction in plant-beneficial rhizobacteria with biocontrol properties.

Biological control of root pathogens--mostly fungi--can be achieved by the introduction of selected bacterial inoculants acting as 'biopesticides'. Successful inoculants have been identified among Gram-negative and Gram-positive bacteria, often belonging to Pseudomonas spp. and Bacillus spp., respectively. Biocontrol activity of a model rhizobacterium, P. fluorescens CHAO, depends to a considerable extent on the synthesis of extracellular antimicrobial secondary metabolites and exoenzymes, thought to antagonize the pathogenicity of a variety of phytopathogenic fungi. The regulation of exoproduct formation in P. fluorescens (as well as in other bacteria) depends essentially on the GacS/GacA two-component system, which activates a largely unknown signal transduction pathway. However, recent evidence indicates that GacS/GacA control has a major impact on target gene expression at a post-transcriptional level, involving an mRNA target sequence (typically near the ribosome binding site), two RNA binding proteins (designated RsmA and RsmE), and a regulatory RNA (RsmZ) capable of binding RsmA. The expression and activity of the regulatory system is stimulated by at least one low-molecular-weight signal. The timing and specificity of this switch from primary to secondary metabolism are essential for effective biocontrol.

Comparative population genetic structure in a plant-pollinator/seed predator system.

Comparative analyses of spatial genetic structure of populations of plants and the insects they interact with provide an indication of how gene flow, natural selection and genetic drift may jointly influence the distribution of genetic variation and potential for local co-adaptation for interacting species. Here, we analysed the spatial scale of genetic structure within and among nine populations of an interacting species pair, the white campion Silene latifolia and the moth Hadena bicruris, along a latitudinal gradient across Northern/Central Europe. This dioecious, short-lived perennial plant inhabits patchy, often disturbed environments. The moth H. bicruris acts both as its pollinator and specialist seed predator that reproduces by laying eggs in S. latifolia flowers. We used nine microsatellite markers for S. latifolia and eight newly developed markers for H. bicruris. We found high levels of inbreeding in most populations of both plant and pollinator/seed predator. Among populations, significant genetic structure was observed for S. latifolia but not for its pollinator/seed predator, suggesting that despite migration among populations of H. bicruris, pollen is not, or only rarely, carried over between populations, thus maintaining genetic structure among plant populations. There was a weak positive correlation between genetic distances of S. latifolia and H. bicruris. These results indicate that while significant structure of S. latifolia populations creates the potential for differentiation at traits relevant for the interaction with the pollinator/seed predator, substantial gene flow in H. bicruris may counteract this process in at least some populations.

Evidence for adaptive radiation from a phylogenetic study of plant defenses.

One signature of adaptive radiation is a high level of trait change early during the diversification process and a plateau toward the end of the radiation. Although the study of the tempo of evolution has historically been the domain of paleontologists, recently developed phylogenetic tools allow for the rigorous examination of trait evolution in a tremendous diversity of organisms. Enemy-driven adaptive radiation was a key prediction of Ehrlich and Raven's coevolutionary hypothesis [Ehrlich PR, Raven PH (1964) Evolution 18:586-608], yet has remained largely untested. Here we examine patterns of trait evolution in 51 North American milkweed species (Asclepias), using maximum likelihood methods. We study 7 traits of the milkweeds, ranging from seed size and foliar physiological traits to defense traits (cardenolides, latex, and trichomes) previously shown to impact herbivores, including the monarch butterfly. We compare the fit of simple random-walk models of trait evolution to models that incorporate stabilizing selection (Ornstein-Ulenbeck process), as well as time-varying rates of trait evolution. Early bursts of trait evolution were implicated for 2 traits, while stabilizing selection was implicated for several others. We further modeled the relationship between trait change and species diversification while allowing rates of trait evolution to vary during the radiation. Species-rich lineages underwent a proportionately greater decline in latex and cardenolides relative to species-poor lineages, and the rate of trait change was most rapid early in the radiation. An interpretation of this result is that reduced investment in defensive traits accelerated diversification, and disproportionately so, early in the adaptive radiation of milkweeds.

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

Linking the population genetics and ecology of arbuscular mycorrhizal fungi

Résumé Les champignons endomycorhiziens arbusculaires (CEA) ont co-évolué avec les plantes terrestres depuis plus de 400 millions d'années. De nos jours, les CEA forment une symbiose avec les racines de la majorité des plantes terrestres. Les CEA sont écologiquement importants parce qu'ils influencent non seulement la croissance des plantes, mais aussi leur diversité. Les CEA sont des biotrophes obligatoires qui reçoivent leur énergie sous forme de glucides issus de la photosynthèse des plantes. En contrepartie, les CEA apportent à leurs hôtes du phospore. Les CEA croissent et se reproduisent clonalement en formant des hyphes et des spores. De plus, les CEA sont coenocytiques et multigénomiques; le cytoplasme d'un CEA contient des noyeaux génétiquement différents. De nombreuses études ont démontré que différentes espèces de CEA agissent différentiellement sur la croissance des plantes. Malgré une conscience de plus en plus forte de l'existence d'une variabilité intraspécifique, la question de savoir si les populations de CEA sont génétiquement variables a été largement négligée. Dans le Chapitre 2, j'ai cherché à savoir si une population de CEA provenant d'un seul champ possède une diversité génétique. Cette étude a mis en évidence une importante variation génétique et phénotypique au sein d'individus de la même population. Des différences au niveau de traits de croissance, héritables et liés à la valeur sélective, indiquent que la variation génétique observée entre isolats n'est pas entièrement neutre. Dans le Chapitre 3, je montre que les différences génétiques entre isolats de CEA d'une population provoquent de la variation dans la croissance des plantes. L'effet des isolats dépend des conditions environnementales et varie de bénéfique à parasitique. Dans le Chapitre 4, je montre que des traits de croissance de CEA varient significativement dans des environnements contrastés. J'ai détecté de fortes interactions entre différents génotypes de CEA et différentes espèces de plantes. Ceci suggère que dans un environnement hétérogène, la sélection pourrait localement favoriser différents génotypes de CEA, maintenant ainsi la diversité génétique dans la population. Les résultats de ce travail aident à mieux comprendre l'importance écologique de la variation intraspécifique des CEA. La possibilité de pouvoir cultiver des individus d'une population de CEA au laboratoire nous a permis une meilleure compréhension de la génétique de ces champignons. De plus, ce travail est une base pour de futures expériences visant à comprendre l'importance évolutive de la diversité intraspécifique des CEA. Abstract Arbuscular mycorrhizal fungi (A1VIF) have co-evolved with land plants -for over 400 million years. Today, AMF form symbioses with roots of most land plants and are ecologically important because they alter plant growth and affect plant diversity. AMF are obligate biotrophs, obtaining their energy in form of plant-derived photosynthates. In return,- they supply their host plants with phosphorous. These fungi grow and reproduce clonally by hyphae and spores. They are coenocytic and multigenomic, harbouring genetically different nuclei in a common cytoplasm. Many studies have shown different AMF species differentially alter plant growth. Despite the increasing awareness of intraspecific variability the question whether there is any genetic variation among different individuals of the same population has been largely neglected. In Chapter 2, we investigated whether there is genetic diversity in a field population of the AMF G. intraradices. This work revealed that large genetic and heritable phenotypic variation exists in this AMF population. Differences in fitness-related growth traits among isolates suggest that some of the observed genetic variation is not selectively neutral. In Chapter 3, we show that genetic differences among isolates from the same population also cause variation in plant growth. The isolate effects on plant growth depended on the environmental conditions and varied from beneficial to detrimental. In Chapter 4, fitnessrelated growth traits of genetically different isolates were significantly altered in contrasting environments. we detected strong AMF isolate by host species interacfions which suggests that in a heterogeneous environment selection could locally favour different AMF genotypes, thereby maintaining high genetic diversity in the population. The results of this work contribute to the understanding of the ecological importance of intraspecific diversity in AMF. The possibility of culturing individuals of an AMF field population under laboratory condition gave new insights into AMF genetics and lays a foundation for future studies to analyse the evolutionary significance of intraspecific genetic diversity in AMF.

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.

Phylogeography and population genetics of north European Atlantic salmon (Salmo salar L.)

Although abundant in the number of individuals, the Atlantic salmon may be considered as a threatened species in many areas of its native distribution range. Human activities such as building of power plant dams, offshore overfishing, pollution, clearing of riverbeds for timber floating and badly designed stocking regimes have diminished the distribution of Atlantic salmon. As a result of this, many of the historical populations both in Europe and northern America have gone extinct or are severely depressed. In fact, only 1% of Atlantic salmon existing today are of natural origin, the rest being farmed salmon. All of this has lead to a vast amount of research and many restoration programmes aiming to bring Atlantic salmon back to rivers from where it has vanished. However, many of the restoration programmes conducted thus far have been unsuccessful due to inadequate scientific research or lack of its implementation, highlighting the fact that more research is needed to fully understand the biology of this complex species. The White and Barents Seas in northwest Russia are among the last regions in Europe where Atlantic salmon populations are still stable, thus forming an important source of biodiversity for the entire European region. Salmon stocks from this area are also of immense economic and social importance for the local people in the form of fishing tourism. The main aim of this thesis was to elucidate the post-glacial history and population genetic structure of north European and particularly northwest Russian Atlantic salmon, both of which are aspects of great importance for the management and conservation of the species. Throughout the whole thesis, these populations were studied by utilizing microsatellites as the main molecular tool. One of the most important discoveries of the thesis was the division of Atlantic salmon from the White and Barents Seas into four separate clusters, which has not been observed in previous studies employing nuclear markers although is supported by mtDNA studies. Populations from the western Barents Sea clustered together with the northeast Atlantic populations into a clearly distinguishable group while populations from the White Sea and eastern Barents Sea were separated into three additional groups. This has important conservation implications as this thesis clearly indicates that conservation of populations from all of the observed clusters is warranted in order to conserve as much of the genetic diversity as possible in this area. The thesis also demonstrates how differences in population life histories within a species, migratory behaviour in this case, and in their phylogeographic origin affect the genetic characteristics of populations, namely diversity and divergence levels. The anadromous populations from the Atlantic Ocean, White Sea and Barents Sea possessed higher levels of genetic diversity than the anadromous populations form the Baltic Sea basin. Among the non-anadromous populations the result was the opposite: the Baltic freshwater populations were more variable. This emphasises the importance of taking the life history of a population into consideration when developing conservation strategies: due to the limited possibilities for new genetic diversity to be generated via gene flow, it is expected that freshwater Atlantic salmon populations would be more vulnerable to extinction following a population crash and thus deserve a high conservation status. In the last chapter of this thesis immune relevant marker loci were developed and screened for signatures of natural selection along with loci linked to genes with other functions or no function at all. Also, a novel landscape genomics method, which combines environmental information with molecular data, was employed to investigate whether immune relevant markers displayed significant correlations to various environmental variables more frequently than other loci. Indications of stronger selection pressure among immune-relevant loci compared to non-immune relevant EST-linked loci was found but further studies are needed to evaluate whether it is a common phenomenon in Atlantic salmon.

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.

Combining niche modelling and landscape genetics to study local adaptation: A novel approach illustrated using alpine plants

Understanding the factors that shape adaptive genetic variation across species niches has become of paramount importance in evolutionary ecology, especially to understand how adaptation to changing climate affects the geographic range of species. The distribution of adaptive alleles in the ecological niche is determined by the emergence of novel mutations, their fitness consequences and gene flow that connects populations across species niches. Striking demographical differences and source sink dynamics of populations between the centre and the margin of the niche can play a major role in the emergence and spread of adaptive alleles. Although some theoretical predictions have long been proposed, the origin and distribution of adaptive alleles within species niches remain untested. In this paper, we propose and discuss a novel empirical approach that combines landscape genetics with species niche modelling, to test whether alleles that confer local adaptation are more likely to occur in either marginal or central populations of species niches. We illustrate this new approach by using a published data set of 21 alpine plant species genotyped with a total of 2483 amplified fragment length polymorphisms (AFLP), distributed over more than 1733 sampling sites across the Alps. Based on the assumption that alleles that were statistically associated with environmental variables were adaptive, we found that adaptive alleles in the margin of a species niche were also present in the niche centre, which suggests that adaptation originates in the niche centre. These findings corroborate models of species range evolution, in which the centre of the niche contributes to the emergence of novel adaptive alleles, which diffuse towards niche margins and facilitate niche and range expansion through subsequent local adaptation. Although these results need to be confirmed via fitness measurements in natural populations and functionally characterised genetic sequences, this study provides a first step towards understanding how adaptive genetic variation emerges and shapes species niches and geographic ranges along environmental gradients.

Why evolution matters for species conservation: perspectives from three case studies of plant metapopulations

We advocate the advantage of an evolutionary approach to conservation biology that considers evolutionary history at various levels of biological organization. We review work on three separate plant taxa, spanning from one to multiple decades, illustrating extremes in metapopulation functioning. We show how the rare endemics Centaurea corymbosa (Clape Massif, France) and Brassica insularis in Corsica (France) may be caught in an evolutionary trap: disruption of metapopulation functioning due to lack of colonization of new sites may have counterselected traits such as dispersal ability or self-compatibility, making these species particularly vulnerable to any disturbance. The third case study concerns the evolution of life history strategies in the highly diverse genus Leucadendron of the South African fynbos. There, fire disturbance and the recolonization phase after fires are so integral to the functioning of populations that recruitment of new individuals is conditioned by fire. We show how past adaptation to different fire regimes and climatic constraints make species with different life history syndromes more or less vulnerable to global changes. These different case studies suggest that management strategies should promote evolutionary potential and evolutionary processes to better protect extant biodiversity and biodiversification.

Genome-wide analysis of adaptive molecular evolution in the carnivorous plant Utricularia gibba

The genome of the bladderwort Utricularia gibba provides an unparalleled opportunity to uncover the adaptive landscape of an aquatic carnivorous plant with unique phenotypic features such as absence of roots, development of water-filled suction bladders, and a highly ramified branching pattern. Despite its tiny size, the U. gibba genome accommodates approximately as many genes as other plant genomes. To examine the relationship between the compactness of its genome and gene turnover, we compared the U. gibba genome with that of four other eudicot species, defining a total of 17,324 gene families (orthogroups). These families were further classified as either 1) lineage-specific expanded/contracted or 2) stable in size. The U. gibba-expanded families are generically related to three main phenotypic features: 1) trap physiology, 2) key plant morphogenetic/developmental pathways, and 3) response to environmental stimuli, including adaptations to life in aquatic environments. Further scans for signatures of protein functional specialization permitted identification of seven candidate genes with amino acid changes putatively fixed by positive Darwinian selection in the U. gibba lineage. The Arabidopsis orthologs of these genes (AXR, UMAMIT41, IGS, TAR2, SOL1, DEG9, and DEG10) are involved in diverse plant biological functions potentially relevant for U. gibba phenotypic diversification, including 1) auxin metabolism and signal transduction, 2) flowering induction and floral meristem transition, 3) root development, and 4) peptidases. Taken together, our results suggest numerous candidate genes and gene families as interesting targets for further experimental confirmation of their functional and adaptive roles in the U. gibba's unique lifestyle and highly specialized body plan.

Concepts in plant disease resistance

Resistance to nearly all pathogens occurs abundantly in our crops. Much of the resistance exploited by breeders is of the major gene type. Polygenic resistance, although used much less, is even more abundantly available. Many types of resistance are highly elusive, the pathogen apparently adapting very easily them. Other types of resistance, the so-called durable resistance, remain effective much longer. The elusive resistance is invariably of the monogenic type and usually of the hypersensitive type directed against specialised pathogens. Race-specificity is not the cause of elusive resistance but the consequence of it. Understanding acquired resistance may open interesting approaches to control pathogens. This is even truer for molecular techniques, which already represent an enourmously wide range of possibilities. Resistance obtained through transformation is often of the quantitative type and may be durable in most cases.

Callus induction and plant regeneration by Brazilian triticale and wheat genotypes

In order to determine the in vitro behavior of Brazilian triticale, 16 triticale genotypes, and three wheat genotypes used as checks, were sown in June 1994. The explants used were immature embryos. In addition to the genotype tests, two culture media for callus induction were also evaluated, i.e., MS (Murashige and Skoog, Physiol. Plant. 15: 473-497, 1962) medium containing 2.0 mg 2,4D/l, and MS medium containing 4.0 mg 2,4D/l. The plant regeneration protocol used was the one employed at the Laboratório de Cultura de Tecidos, Departamento de Plantas de Lavoura, Universidade Federal do Rio Grande do Sul, for wheat. Differences in plant regeneration were observed both among triticale and wheat genotypes, with triticale usually showing better regeneration than wheat. No differences were observed between the callus induction media.

Plant height reduction in populations of triticale (X triticosecale Wittmack) by induced mutations and artificial crosses

Induced mutations by gamma radiation (0, 5, 10, 20 and 40 kR doses) and reciprocal crosses were tested as mechanisms of enhancing genetic variability for plant height in two triticale cultivars, BR4 and EMBRAPA18. The reciprocal crosses and all doses of radiation showed similar increase in genetic amplitude for this trait, being suitable for increasing variability in breeding programs. Genotypes showed different responses as the gamma ray doses were increased, expressing shorter plant height. The decision of using induced mutations or artificial crosses depends on the resources available and the selection method to be used