Caracterização fisiológica e do perfil de expressão gênica do transporte de nitrogênio em genótipos contrastantes para processo de fixação biológica de N2 de cana-de-açúcar (Saccharum spp.)

A cana-de-açúcar é uma cultura agrícola de grande importância econômica para o Brasil, e a expansão de seu cultivo para solos marginais requer uma maior utilização de fertilizantes à base de nitrogênio (N). Na maioria dos países produtores, a adubação nitrogenada se baseia em altas doses de aplicação, enquanto, no Brasil, o seu uso é relativamente baixo devido, em parte, ao processo de fixação biológica de nitrogênio (FBN) pela ação de bactérias diazotróficas. Além da FBN, as plantas adquirem fontes de N, como amônio e nitrato, por meio de transportadores de membranas localizados nas raízes. Há evidências que a associação com microrganismos pode favorecer as plantas por meio da regulação dos genes de transportadores de N. Desta forma, este trabalho teve como objetivo caracterizar o transporte de amônio e nitrato, avaliando a expressão gênica dos principais transportadores de N em cana-de-açúcar cultivada in vitro sob o efeito da associação com bactérias diazotróficas. Também foi descrita a comunidade bacteriana de plântulas in vitro, bem como o efeito da fertilização com N e da inoculação com bactérias diazotróficas em plantas maduras. Plântulas de \'SP70- 1143\' e \'Chunee\', que contrastam para FBN, foram empregadas em ensaios in vitro sob diversas concentrações e fontes de N em associação ou não com uma estirpe de Gluconacetobacter diazotrophicus ou um mistura de bactérias diazotróficas (G. diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicans, Azospirillum amazonense e Burkholderia tropica). A caracterização do transporte de N por meio de ensaios de absorção de nitrato e amônio marcados (15N) revelou que a interação entre cana-de-açúcar x G. diazotrophicus induziu a expressão do gene do transportador de nitrato ScNRT2.1, o que levou a uma tendência no aumento no influxo de nitrato, assim como dos genes de transportadores de amônio ScAMT1.1 e ScAMT1.3, resultando em maiores influxos de amônio apenas para a cultivar \'SP70- 1143\'. Já a associação da cana-de-açúcar com a mistura de bactérias diazotróficas revelou que somente houve indução transcricional de ScAMT1.1, o que resultou na maior absorção de amônio em \'SP70-1143\'. Por sua vez, quando analisada a interação in vitro por 30 dias, a presença da bactéria, apesar de transiente, possivelmente favoreceu a expressão dos genes de transportadores de nitrato ScNRT1.1 e ScNRT2.1, e do transportador de amônio ScAMT1.1, resultando no maior acúmulo de 15N-nitrato de amônio nas plantas de \'SP70-1143\'. Foi detectada uma comunidade bacteriana associada a plântulas micropropagadas, a qual é distinta entre os genótipos \'SP70-1143\' e \'Chunee\' e se altera com a inoculação com G. diazotrophicus. Para as plantas cultivadas em campo, a comunidade bacteriana existente foi alterada pela fertilização de N, mas não pela inoculação com diazotróficas. Portanto, a inoculação com bactérias diazotróficas parece induzir a expressão dos principais genes transportadores de amônio e nitrato em plântulas do genótipo \'SP70-1143\' resultando na maior absorção de fontes inorgânicas de N.

Análise de metabólitos e proteínas totais em folhas de Eucalyptus grandis durante a infecção por Puccinia psidii

Os mecanismos moleculares envolvidos na resistência de plantas contra patógenos são um tema bastante discutido no meio acadêmico, sendo o objetivo maior dos estudos a diminuição das perdas de produtividade provocadas por doenças em plantações do mundo todo. Muitos modelos de interação patógeno-hospedeiro foram propostos e desenvolvidos priorizando plantas e culturas de rápido desenvolvimento com ciclo de vida curto. Espécies de ciclo longo, porém, devem lidar durante anos - ao menos até a idade reprodutiva - contra o ataque de bactérias, fungos e vírus, sem contar, nesse meio tempo, com recombinações genéticas e mutações que tornariam possível o escape contra as moléstias causadas por microrganismos. Assim, como alternativa aos modelos usuais, o presente trabalho estudou um diferente par de antagonistas: Eucalyptus grandis e Puccinia psidii. Apesar da contribuição de programas de melhoramento genético, o patossistema E. grandis X P. psidii ainda é pouco descrito no nível molecular, havendo poucos estudos sobre os processos e as moléculas que agem de forma a conferir resistência às plantas. Assim, buscando o melhor entendimento da relação entre E. grandis X P. psidii, o presente trabalho estudou a mudança dos perfis de proteínas e metabólitos secundários ocorrida nos tecidos foliares de plantas resistentes e susceptíveis durante a infecção pelo patógeno, com o auxílio da técnica de cromatografia líquida acoplada à espectrometria de massas. Os resultados obtidos indicam que as plantas resistentes percebem a presença do patógeno logo nas primeiras horas pós-infecção, produzindo proteínas ligadas à imunidade (HSP90, ILITYHIA, LRR Kinase, NB-ARC disease resistance protein). Essa percepção desencadeia a produção de proteínas de parede celular e de resposta oxidativa, além de modificar o metabolismo primário e secundário. As plantas susceptíveis, por outro lado, têm o metabolismo subvertido, produzindo proteínas responsáveis pelo afrouxamento da parede celular, beneficiando a absorção de nutrientes, crescimento e propagação de P. psidii. No trabalho também são propostos metabólitos biomarcadores de resistência, moléculas biomarcadoras de resposta imune e sinais da infecção por patógeno em E. grandis.

Learning, odour preference and flower foraging in moths

Floral volatiles play a major role in plant-insect communication. We examined the influence of two volatiles, phenylacetaldehyde and a-pinene, on the innate and learnt foraging behaviour of the moth Helicoverpa armigera. In dual-choice wind tunnel tests, adult moths flew upwind towards both volatiles, with a preference for phenylacetaldehyde. When exposure to either of these volatiles was paired with a feeding stimulus (sucrose), all moths preferred the learnt odour in the preference test. This change in preference was not seen when moths were exposed to the odour without a feeding stimulus. The learnt preference for the odour was reduced when moths were left unfed for 24 h before the preference test. We tested whether moths could discriminate between flowers that differed in a single volatile component. Moths were trained to feed on flowers that were odour-enhanced using either phenylacetaldehyde or a-pinene. Choice tests were then carried out in an outdoor flight cage, using flowers enhanced with either volatile. Moths showed a significant preference for the flower type on which they were trained. Moths that were conditioned on flowers that were not odour-enhanced showed no preference for either of the odour-enhanced flower types. The results imply that moths may be discriminating among odour profiles of individual flowers from the same species. We discuss this behaviour within the context of nectar foraging in moths and odour signalling by flowering plants.

Rhizodeposits of Trifolium pratense and Lolium perenne: Their comparative effects on 2,4-D mineralization in two contrasting soils

Rhizosphere enhanced biodegradation of organic pollutants has been reported frequently and a stimulatory role for specific components of rhizodeposits postulated. As rhizodeposit composition is a function of plant species and soil type, we compared the effect of Lolium perenne and Trifolium pratense grown in two different soils (a sandy silt loam: pH 4, 2.8% OC, no previous 2,4-D exposure and a silt loam: pH 6.5, 4.3% OC, previous 2,4-D exposure) on the mineralization of the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid). We investigated the relationship of mineralization kinetics to dehydrogenase activity, most probable number of 2,4-D degraders (MPN2,4-D) and 2,4-D degrader composition (using sequence analysis of the gene encoding alpha-ketoglutarate/2,4-D dioxygenase (tfdA)). There were significant (P < 0.01) plant-soil interaction effects on MPN2,4-D and 2,4-D mineralization kinetics (e.g. T pratense rhizodeposits enhanced the maximum mineralization rate by 30% in the acid sandy silt loam soil, but not in the neutral silt loam soil). Differences in mineralization kinetics could not be ascribed to 2,4-D degrader composition as both soils had tfdA sequences which clustered with tfdAs representative of two distinct classes of 2,4-D degrader: canonical R. eutropha JMP134-like and oligotrophic alpha-proteobacterial-like. Other explanations for the differential rhizodeposit effect between soils and plants (e.g. nutrient competition effects) are discussed. Our findings stress that complexity of soil-plant-microbe interactions in the rhizosphere make the occurrence and extent of rhizosphere-enhanced xenobiotic degradation difficult to predict.

Ecology of Leptocoris Hahn (Hemiptera: Rhopalidae) Soapberry Bugs in Australia

Soapberry bugs are worldwide seed predators of plants in the family Sapindaceae. Australian sapinds are diverse and widespread, consisting of about 200 native trees and shrubs. This flora also includes two introduced environmental weeds, plus cultivated lychee (Litchi chinensis Sonn.), longan (Dimocarpus longan Lour.) and rambutan (Nephelium lappaceum L.). Accordingly, Australian soapberry bugs may be significant in ecology, conservation and agriculture. Here we provide the first account of their ecology. We find five species of Leptocoris Hahn in Australia, and list sapinds that do and do not serve as reproductive hosts. From museum and field records we map the continental distributions of the insects and primary hosts. Frequency of occupation varies among host species, and the number of hosts varies among the insects. In addition, differences in body size and beak length are related to host use. For example, the long-beaked Leptocoris tagalicus Burmeister is highly polyphagous in eastern rainforests, where it occurs on at least 10 native and non-native hosts. It aggregates on hosts with immature fruit and commences feeding before fruits dehisce. Most of its continental range, however, matches that of a single dryland tree, Atalaya hemiglauca F. Muell., which has comparatively unprotected seeds. The taxon includes a smaller and shorter-beaked form that is closely associated with Atalaya, and appears to be taxonomically distinct. The other widespread soapberry bug is the endemic Leptocoris mitellatus Bergroth. It too is short-beaked, and colonises hosts phenologically later than L. tagalicus, as seeds become more accessible in open capsules. Continentally its distribution is more southerly and corresponds mainly to that of Alectryon oleifolius Desf. Among all host species, the non-native environmental weeds Cardiospermum L. and Koelreuteria Laxm. are most consistently attacked, principally by L. tagalicus. These recent host shifts have biocontrol implications. In contrast, the sapinds planted as fruit crops appear to be less frequently used at present and mainly by the longer-beaked species.

Climate Change or Urbanization? Impacts on a Traditional Coffee Production System in East Africa over the Last 80 Years

Global environmental changes (GEC) such as climate change (CC) and climate variability have serious impacts in the tropics, particularly in Africa. These are compounded by changes in land use/land cover, which in turn are driven mainly by economic and population growth, and urbanization. These factors create a feedback loop, which affects ecosystems and particularly ecosystem services, for example plant-insect interactions, and by consequence agricultural productivity. We studied effects of GEC at a local level, using a traditional coffee production area in greater Nairobi, Kenya. We chose coffee, the most valuable agricultural commodity worldwide, as it generates income for 100 million people, mainly in the developing world. Using the coffee berry borer, the most serious biotic threat to global coffee production, we show how environmental changes and different production systems (shaded and sun-grown coffee) can affect the crop. We combined detailed entomological assessments with historic climate records (from 1929-2011), and spatial and demographic data, to assess GEC's impact on coffee at a local scale. Additionally, we tested the utility of an adaptation strategy that is simple and easy to implement. Our results show that while interactions between CC and migration/urbanization, with its resultant landscape modifications, create a feedback loop whereby agroecosystems such as coffee are adversely affected, bio-diverse shaded coffee proved far more resilient and productive than coffee grown in monoculture, and was significantly less harmed by its insect pest. Thus, a relatively simple strategy such as shading coffee can tremendously improve resilience of agro-ecosystems, providing small-scale farmers in Africa with an easily implemented tool to safeguard their livelihoods in a changing climate.

Pre-announcement of symbiotic guests: transcriptional reprogramming by mycorrhizal lipochitooligosaccharides shows a strict co-dependency on the GRAS transcription factors NSP1 and RAM1

BACKGROUND: More than 80 % of all terrestrial plant species establish an arbuscular mycorrhiza (AM) symbiosis with Glomeromycota fungi. This plant-microbe interaction primarily improves phosphate uptake, but also supports nitrogen, mineral, and water aquisition. During the pre-contact stage, the AM symbiosis is controled by an exchange of diffusible factors from either partner. Amongst others, fungal signals were identified as a mix of sulfated and non-sulfated lipochitooligosaccharides (LCOs), being structurally related to rhizobial nodulation (Nod)-factor LCOs that in legumes induce the formation of nitrogen-fixing root nodules. LCO signals are transduced via a common symbiotic signaling pathway (CSSP) that activates a group of GRAS transcription factors (TFs). Using complex gene expression fingerprints as molecular phenotypes, this study primarily intended to shed light on the importance of the GRAS TFs NSP1 and RAM1 for LCO-activated gene expression during pre-symbiotic signaling. RESULTS: We investigated the genome-wide transcriptional responses in 5 days old primary roots of the Medicago truncatula wild type and four symbiotic mutants to a 6 h challenge with LCO signals supplied at 10(-7/-8) M. We were able to show that during the pre-symbiotic stage, sulfated Myc-, non-sulfated Myc-, and Nod-LCO-activated gene expression almost exclusively depends on the LysM receptor kinase NFP and is largely controled by the CSSP, although responses independent of this pathway exist. Our results show that downstream of the CSSP, gene expression activation by Myc-LCOs supplied at 10(-7/-8) M strictly required both the GRAS transcription factors RAM1 and NSP1, whereas those genes either co- or specifically activated by Nod-LCOs displayed a preferential NSP1-dependency. RAM1, a central regulator of root colonization by AM fungi, controled genes activated by non-sulfated Myc-LCOs during the pre-symbiotic stage that are also up-regulated in areas with early physical contact, e.g. hyphopodia and infecting hyphae; linking responses to externally applied LCOs with early root colonization. CONCLUSIONS: Since both RAM1 and NSP1 were essential for the pre-symbiotic transcriptional reprogramming by Myc-LCOs, we propose that downstream of the CSSP, these GRAS transcription factors act synergistically in the transduction of those diffusible signals that pre-announce the presence of symbiotic fungi.

Challenges in imaging and predictive modeling of rhizosphere processes

Background Plant-soil interaction is central to human food production and ecosystem function. Thus, it is essential to not only understand, but also to develop predictive mathematical models which can be used to assess how climate and soil management practices will affect these interactions. Scope In this paper we review the current developments in structural and chemical imaging of rhizosphere processes within the context of multiscale mathematical image based modeling. We outline areas that need more research and areas which would benefit from more detailed understanding. Conclusions We conclude that the combination of structural and chemical imaging with modeling is an incredibly powerful tool which is fundamental for understanding how plant roots interact with soil. We emphasize the need for more researchers to be attracted to this area that is so fertile for future discoveries. Finally, model building must go hand in hand with experiments. In particular, there is a real need to integrate rhizosphere structural and chemical imaging with modeling for better understanding of the rhizosphere processes leading to models which explicitly account for pore scale processes.

Climate oscillations and species interactions: large-scale congruence but regional differences in the phylogeographic structures of an alpine plant and its monophagous insect

Aim. To predict the fate of alpine interactions involving specialized species, using a monophagous beetle and its host-plant as a case study. Location. The Alps. Methods. We investigated genetic structuring of the herbivorous beetle Oreina gloriosa and its specific host-plant Peucedanum ostruthium. We used genome fingerprinting (in the insect and the plant) and sequence data (in the insect) to compare the distribution of the main gene pools in the two associated species and to estimate divergence time in the insect, a proxy for the temporal origin of the interaction. We quantified the similarity in spatial genetic structures by performing a Procrustes analysis, a tool from the shape theory. Finally, we simulated recolonization of an empty space analogous to the deglaciated Alps just after ice retreat by two lineages from two species showing unbalanced dependence, to examine how timing of the recolonization process, as well as dispersal capacities of associated species, could explain the observed pattern. Results. Contrasting with expectations based on their asymmetrical dependence, patterns in the beetle and plant were congruent at a large scale. Exceptions occurred at a regional scale in areas of admixture, matching known suture zones in Alpine plants. Simulations using a lattice-based model suggested these empirical patterns arose during or soon after recolonization, long after the estimated origin of the interaction c. 0.5 million years ago. Main conclusions. Species-specific interactions are scarce in alpine habitats because glacial cycles have limited opportunities for coevolution. Their fate, however, remains uncertain under climate change. Here we show that whereas most dispersal routes are paralleled at large scale, regional incongruence implies that the destinies of the species might differ under changing climate. This may be a consequence of the host-dependence of the beetle that locally limits the establishment of dispersing insects.

Molecular basis and regulation of insect pathogenicity in plant-beneficial pseudomonads

Les bactéries du genre Pseudomonas ont la capacité étonnante de s'adapter à différents habitats et d'y survivre, ce qui leur a permis de conquérir un large éventail de niches écologiques et d'interagir avec différents organismes hôte. Les espèces du groupe Pseudomonas fluorescens peuvent être facilement isolées de la rhizosphère et sont communément connues comme des Pseudomonas bénéfiques pour les plantes. Elles sont capables d'induire la résistance systémique des plantes, d'induire leur croissance et de contrer des phytopathogènes du sol. Un sous-groupe de ces Pseudomonas a de plus développé la capacité d'infecter et de tuer certaines espèces d'insectes. Approfondir les connaissances sur l'interaction de ces bactéries avec les insectes pourraient conduire au développement de nouveaux biopesticides pour la protection des cultures. Le but de cette thèse est donc de mieux comprendre la base moléculaire, l'évolution et la régulation de la pathogénicité des Pseudomonas plante-bénéfiques envers les insectes. Plus spécifiquement, ce travail a été orienté sur l'étude de la production de la toxine insecticide appelée Fit et sur l'indentification d'autres facteurs de virulence participant à la toxicité de la bactérie envers les insectes. Dans la première partie de ce travail, la régulation de la production de la toxine Fit a été évaluée par microscopie à épifluorescence en utilisant des souches rapportrices de Pseudomonas protegens CHA0 qui expriment la toxine insecticide fusionnée à une protéine fluorescente rouge, au site natif du gène de la toxine. Celle-ci a été détectée uniquement dans l'hémolymphe des insectes et pas sur les racines des plantes, ni dans les milieux de laboratoire standards, indiquant une production dépendante de l'hôte. L'activation de la production de la toxine est contrôlée par trois protéines régulatrices dont l'histidine kinase FitF, essentielle pour un contrôle précis de l'expression et possédant un domaine "senseur" similaire à celui de la kinase DctB qui régule l'absorption de carbone chez les Protéobactéries. Il est donc probable que, durant l'évolution de FitF, un réarrangement de ce domaine "senseur" largement répandu ait contribué à une production hôte-spécifique de la toxine. Les résultats de cette étude suggèrent aussi que l'expression de la toxine Fit est plutôt réprimée en présence de composés dérivés des plantes qu'induite par la perception d'un signal d'insecte spécifique. Dans la deuxième partie de ce travail, des souches mutantes ciblant des facteurs de virulence importants identifiés dans des pathogènes connus ont été générées, dans le but d'identifier ceux avec une virulence envers les insectes atténuée. Les résultats ont suggéré que l'antigène O du lipopolysaccharide (LPS) et le système régulateur à deux composantes PhoP/PhoQ contribuent significativement à la virulence de P. protegens CHA0. La base génétique de la biosynthèse de l'antigène O dans les Pseudomonas plante-bénéfiques et avec une activité insecticide a été élucidée et a révélé des différences considérables entre les lignées suite à des pertes de gènes ou des acquisitions de gènes par transfert horizontal durant l'évolution de certaines souches. Les chaînes latérales du LPS ont été montrées comme vitales pour une infection des insectes réussie par la souche CHA0, après ingestion ou injection. Les Pseudomonas plante-bénéfiques, avec une activité insecticide sont naturellement résistants à la polymyxine B, un peptide antimicrobien modèle. La protection contre ce composé antimicrobien particulier dépend de la présence de l'antigène O et de la modification du lipide A, une partie du LPS, avec du 4-aminoarabinose. Comme les peptides antimicrobiens cationiques jouent un rôle important dans le système immunitaire des insectes, l'antigène O pourrait être important chez les Pseudomonas insecticides pour surmonter les mécanismes de défense de l'hôte. Le système PhoP/PhoQ, connu pour contrôler les modifications du lipide A chez plusieurs bactéries pathogènes, a été identifié chez Pseudomonas chlororaphis PCL1391 et P. protegens CHA0. Pour l'instant, il n'y a pas d'évidence que des modifications du lipide A contribuent à la pathogénicité de cette bactérie envers les insectes. Cependant, le senseur-kinase PhoQ est requis pour une virulence optimale de la souche CHA0, ce qui suggère qu'il régule aussi l'expression des facteurs de virulence de cette bactérie. Les découvertes de cette thèse démontrent que certains Pseudomonas associés aux plantes sont de véritables pathogènes d'insectes et donnent quelques indices sur l'évolution de ces microbes pour survivre dans l'insecte-hôte et éventuellement le tuer. Les résultats suggèrent également qu'une recherche plus approfondie est nécessaire pour comprendre comment ces bactéries sont capables de contourner ou surmonter la réponse immunitaire de l'hôte et de briser les barrières physiques pour envahir l'insecte lors d'une infection orale. Pour cela, les futures études ne devraient pas uniquement se concentrer sur le côté bactérien de l'interaction hôte-microbe, mais aussi étudier l'infection du point de vue de l'hôte. Les connaissances gagnées sur la pathogénicité envers les insectes des Pseudomonas plante-bénéfiques donnent un espoir pour une future application en agriculture, pour protéger les plantes, non seulement contre les maladies, mais aussi contre les insectes ravageurs. -- Pseudomonas bacteria have the astonishing ability to survive within and adapt to different habitats, which has allowed them to conquer a wide range of ecological niches and to interact with different host organisms. Species of the Pseudomonas fluorescens group can readily be isolated from plant roots and are commonly known as plant-beneficial pseudomonads. They are capable of promoting plant growth, inducing systemic resistance in the plant host and antagonizing soil-borne phytopathogens. A defined subgroup of these pseudomonads evolved in addition the ability to infect and kill certain insect species. Profound knowledge about the interaction of these particular bacteria with insects could lead to the development of novel biopesticides for crop protection. This thesis thus aimed at a better understanding of the molecular basis, evolution and regulation of insect pathogenicity in plant-beneficial pseudomonads. More specifically, it was outlined to investigate the production of an insecticidal toxin termed Fit and to identify additional factors contributing to the entomopathogenicity of the bacteria. In the first part of this work, the regulation of Fit toxin production was probed by epifluorescence microscopy using reporter strains of Pseudomonas protegens CHAO that express a fusion between the insecticidal toxin and a red fluorescent protein in place of the native toxin gene. The bacterium was found to express its insecticidal toxin only in insect hemolymph but not on plant roots or in common laboratory media. The host-dependent activation of Fit toxin production is controlled by three local regulatory proteins. The histidine kinase of this regulatory system, FitF, is essential for the tight control of toxin expression and shares a sensing domain with DctB, a sensor kinase regulating carbon uptake in Proteobacteria. It is therefore likely that shuffling of a ubiquitous sensor domain during the evolution of FitF contributed to host- specific production of the Fit toxin. Findings of this study additionally suggest that host-specific expression of the Fit toxin is mainly achieved by repression in the presence of plant-derived compounds rather than by induction upon perceiving an insect-specific signal molecule. In the second part of this thesis, mutant strains were generated that lack factors previously shown to be important for virulence in prominent pathogens. A screening for attenuation in insect virulence suggested that lipopolysaccharide (LPS) O-antigen and the PhoP-PhoQ two-component regulatory system significantly contribute to virulence of P. protegens CHAO. The genetic basis of O-antigen biosynthesis in plant-beneficial pseudomonads displaying insect pathogenicity was elucidated and revealed extensive differences between lineages due to reduction and horizontal acquisition of gene clusters during the evolution of several strains. Specific 0 side chains of LPS were found to be vital for strain CHAO to successfully infect insects by ingestion or upon injection. Insecticidal pseudomonads with plant-beneficial properties were observed to be naturally resistant to polymyxin B, a model antimicrobial peptide. Protection against this particular antimicrobial compound was dependent on the presence of O-antigen and modification of the lipid A portion of LPS with 4-aminoarabinose. Since cationic antimicrobial peptides play a major role in the immune system of insects, O-antigenic polysaccharides could be important for insecticidal pseudomonads to overcome host defense mechanisms. The PhoP-PhoQ system, which is well-known to control lipid A modifications in several pathogenic bacteria, was identified in Pseudomonas chlororaphis PCL1391 and P. protegens CHAO. No evidence was found so far that lipid A modifications contribute to insect pathogenicity in this bacterium. However, the sensor kinase PhoQ was required for full virulence of strain CHAO suggesting that it additionally regulates the expression of virulence factors in this bacterium. The findings of this thesis demonstrate that certain plant-associated pseudomonads are true insect pathogens and give some insights into how these microbes evolved to survive within and eventually kill the insect host. Results however also point out that more in-depth research is needed to know how exactly these fascinating bacteria manage to bypass or overcome host immune responses and to breach physical barriers to invade insects upon oral infection. To achieve this, future studies should not only focus on the bacterial side of the microbe-host interactions but also investigate the infection from a host-oriented view. The knowledge gained about the entomopathogenicity of plant-beneficial pseudomonads gives hope for their future application in agriculture to protect plants not only against plant diseases but also against insect pests.

Mutualism from the inside: coordinated development of plant and insect in an active pollinating fig wasp

Recent studies on the obligate interaction between fig trees and their pollinating agaonid wasps have focused on population aspects and wasp-seed exploitation at the level of the inflorescence. Detailed studies on larval and gall development are required to more fully understand how resources are exploited and adaptations fine-tuned by each partner in nursery pollination mutualisms. We studied the larval development of the active pollinating fig wasp, Pegoscapus sp., and the galling process of individual flowers within the figs of its monoecious host, Ficus citrifolia, in Brazil. The pollinator development is strongly dependent on flower pollination. Figs entered by pollen-free wasps were in general more likely to abort. Retained, unpollinated figs had both higher larval mortality and a lower number of wasps. Pegoscapus sp. larvae are adapted to plant development, with two contrasting larval feeding strategies proceeding alongside gall development. The first two larval stages behave as ovary parasites. Later larval stages feed on hypertrophied endosperm. This indicates that a successful galling process relies on endosperm, and also reveals why pollination would be a prerequisite for the production of high-quality galls for this Pegoscapus species.

Chemical phenotype matching between a plant and its insect herbivore

Two potential outcomes of a coevolutionary interaction are an escalating arms race and stable cycling. The general expectation has been that arms races predominate in cases of polygenic inheritance of resistance traits and permanent cycling predominates in cases in which resistance is controlled by major genes. In the interaction between Depressaria pastinacella, the parsnip webworm, and Pastinaca sativa, the wild parsnip, traits for plant resistance to insect herbivory (production of defensive furanocoumarins) as well as traits for herbivore “virulence” (ability to metabolize furanocoumarins) are characterized by continuous heritable variation. Furanocoumarin production in plants and rates of metabolism in insects were compared among four midwestern populations; these traits then were classified into four clusters describing multitrait phenotypes occurring in all or most of the populations. When the frequency of plant phenotypes belonging to each of the clusters is compared with the frequency of the insect phenotypes in each of the clusters across populations, a remarkable degree of frequency matching is revealed in three of the populations. That frequencies of phenotypes vary among populations is consistent with the fact that spatial variation occurs in the temporal cycling of phenotypes; such processes contribute in generating a geographic mosaic in this coevolutionary interaction on the landscape scale. Comparisons of contemporary plant phenotype distributions with phenotypes of herbarium specimens collected 9–125 years ago from across a similar latitudinal gradient, however, suggest that for at least one resistance trait—sphondin concentration—interactions with webworms have led to escalatory change.

The aphid transmission factor of cauliflower mosaic virus forms a stable complex with microtubules in both insect and plant cells

We analyzed the distribution of the cauliflower mosaic virus (CaMV) aphid transmission factor (ATF), produced via a baculovirus recombinant, within Sf9 insect cells. Immunogold labeling revealed that the ATF colocalizes with an atypical cytoskeletal network. Detailed observation by electron microscopy demonstrated that this network was composed of microtubules decorated with paracrystalline formations, characteristic of the CaMV ATF. A derivative mutant of the ATF, unable to self-assemble into paracrystals, was also analyzed. This mutant formed a net-like structure, with a mesh of four nanometers, tightly sheathing microtubules. Both the ATF– and the derivative mutant–microtubule complexes were highly stable. They resisted dilution-, cold-, and calcium-induced microtubule disassembly as well as a combination of all three for over 6 hr. CaMV ATF cosedimented with microtubules and, surprisingly, it bound to Taxol-stabilized microtubules at high ionic strength, thus suggesting an atypical interaction when compared with that usually described for microtubule-binding proteins. Using immunofluorescence double labeling we also demonstrated that the CaMV ATF colocalizes with the microtubule network when expressed in plant cells.

Oviposition by a moth suppresses constitutive and herbivore-induced plant volatiles in maize

Plant volatiles function as important signals for herbivores, parasitoids, predators, and neighboring plants. Herbivore attack can dramatically increase plant volatile emissions in many species. However, plants do not only react to herbivore-inflicted damage, but also already start adjusting their metabolism upon egg deposition by insects. Several studies have found evidence that egg deposition itself can induce the release of volatiles, but little is known about the effects of oviposition on the volatiles released in response to subsequent herbivory. To study this we measured the effect of oviposition by Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) moths on constitutive and herbivore-induced volatiles in maize (Zea mays L.). Results demonstrate that egg deposition reduces the constitutive emission of volatiles and suppresses the typical burst of inducible volatiles following mechanical damage and application of caterpillar regurgitant, a treatment that mimics herbivory. We discuss the possible mechanisms responsible for reducing the plant`s signaling capacity triggered by S. frugiperda oviposition and how suppression of volatile organic compounds can influence the interaction between the plant, the herbivore, and other organisms in its environment. Future studies should consider oviposition as a potential modulator of plant responses to insect herbivores.