Evaluation of alfalfa resistance to the pea aphid, Acyrtosiphon pisum [Homoptera : Aphididae] – Methodological aspects to improve a standardized speedling test

Cette étude concerne des éléments de standardisation pour mettre en oeuvre un test d'évaluation de la résistance variétale de la luzerne (Medicago sativa) au puceron du pois (Acyrthosiphon pisum). Réalisé en conditions contrôlées, il évalue la résistance de plantules à l'infestation aphidienne. Pour réaliser cette dernière, il est nécessaire de produire les pucerons sur des plantes de luzerne plutôt que sur des plantes de féverole, leur action sur la luzerne étant alors moindre. La disposition d'un seul cultivar (condition de «non choix») donne les mêmes résultats que la disposition dans la même unité de plusieurs variétés (conditions de choix) et est plus facile à gérer. L'unité élémentaire, constituée de 54 plantules, est infestée avec 360 mg de pucerons lorsque les cotylédons s'ouvrent, ce que l'on renouvelle après 5 jours. Cette dose conduit à des résultats optimums par rapport à des infestations réalisées dans les mêmes conditions avec 180 ou 540 mg. L'infestation est arrêtée lorsque 60 % des plantules d'un cultivar sensible dépérissent ou meurent. Dans ces conditions, nous avons calculé le nombre de répétitions nécessaires. Six unités élémentaires permettent alors d'apprécier une différence de mortalité entre cultivars de 20 %.This study proposes a guide for the design of experiments to test alfalfa (Medicago sativa) for resistance to pea aphid infestation (Acyrthosiphon pisum). This test was conducted in controlled conditions on alfalfa seedlings. For the infestation, aphid population maintained on alfalfa was found to be more efficient than an aphid population reared on broad bean. When comparing alfalfa cultivars, a non-choice test gave the same results as a choice test, that was more difficult to perform. When infesting a unit of 54 seedlings at the cotyledon stage on the 1st and 5th day of the experiment, 360 mg compared with 180 mg and 540 mg aphids, led to the best compromise between levels of infestation and aphid stock culture availability. Infestation was stopped when more than 60% of susceptible cultivar seedlings were wilted or dead. Under these conditions, we calculated the number of replicates necessary to obtain a fixed level of difference. Six units per cultivar would distinguish between cultivars differing from 20% in their seedling mortality

Contribution à l'étude de la régulation de l'expression du gène humain UCP3 dans le muscle squelettique de souris : étude expérimentale

UCP3 est une protéine découplante de la membrane interne mitochondriale exclusivement exprimée ou presque dans les muscles squelettiques chez l’Homme et potentiellement impliquée dans le métabolisme des acides gras. Ce travail concerne la régulation de l’expression du gène humain UCP3 chez la souris. Des animaux portant le gène UCP3 en entier ou en partie ont été créés par transgenèse additive dans le but de délimiter les séquences responsables de la spécificité musculaire d’expression et celles responsables de la réponse aux nutriments. Une séquence intronique de 600 pb confère l’expression musculaire in vivo. Le jeûne et le sevrage sont deux situations entraînant une induction de l’expression du transgène et de la forme murine d’UCP3. Ces manipulations nutritionnelles font intervenir les acides gras, modulateurs connus de l’expression d’UCP3. Cependant, d’autres facteurs de régulation pourraient être impliqués dans la régulation de l’expression du gène UCP3

Régulation de la protéine découplante UCP3 et inhibition génique ou pharmacologique de la lipase hormono-sensible

Ce manuscrit est premièrement axé sur la régulation de l'expression de la protéine découplante 3 (UCP3). UCP3 est une protéine découplante de la membrane interne mitochondriale exclusivement exprimée ou presque dans les muscles squelettiques chez l'Homme et potentiellement impliquée dans le métabolisme des acides gras. Des souris portant le gène UCP3 humain entier ou en partie ont été créés par transgenèse additive dans le but de délimiter les séquences responsables de la spécificité musculaire d'expression. Nous avons identifié une séquence intronique de 600 pb qui confère l'expression musculaire in vivo. Dans une deuxième partie, une étude des conséquences fonctionnelles d'une altération des capacités lipolytiques du tissu adipeux a été réalisée chez la souris. La lipolyse adipocytaire est en partie assurée par la lipase hormono-sensible (LHS). L'expression et l'activité de la LHS sont diminuées dans le tissu adipeux du sujet obèse et/ou insulino-résistant. Nous avons développé des modèles animaux d'inhibition génique et pharmacologique de la LHS chez lesquels nous avons caractérisé le métabolisme énergétique, l'insulino-sensibilité et l'inflammation du tissu adipeux. La diminution des capacités lipolytiques est accompagnée d'une amélioration de la sensibilité à l'insuline et d’une modification du métabolisme des acides gras dans ces deux modèles. L'inflammation du tissu adipeux, reconnue comme facteur modulateur de l'insulino-sensibilité, ne semble pas être impliquée dans ce phénotype.In a first part, this study focuses on the regulation of the expression of the uncoupling protein-3 (UCP3). UCP3 is an inner mitochondrial protein almost exclusively expressed in skeletal muscle in human which could be implicated in fatty acid metabolism. A transgenesis approach has been used to create animal bearing all or part of the UCP3 gene in order to delineate the sequences responsible for the muscle specific expression. A 600 bp sequence of the human UCP3 intron 1 gene has been identified which confers the muscular expression in vivo. The second part of this study is dedicated to the functional consequences of the alteration of lipolytic capacities in mouse adipose tissue. Adipocyte lipolysis is partly achieved by the hormone sensitive-lipase (HSL). HSL expression and activity is altered in adipose tissue of obese and/or insulin resistant subjects. We developed mouse models of genetic and pharmacological inhibition of HSL. Reduction of lipolytic capacity was associated with an improvement of insulin sensitivity and fatty acid metabolism in both animal models. Adipose tissue inflammation, that is a well known modulator of insulin sensitivity, did not seem to be involved in this phenotype

Phloem sap intricacy and interplay with aphid feeding

Aphididae feed upon the plant sieve elements (SE), where they ingest sugars, nitrogen compounds and other nutrients. For ingestion, aphid stylets penetrate SE, and because of the high hydrostatic pressure in SE, phloem sap exudes out into the stylets. Severing stylets to sample phloem exudates (i.e. stylectomy) has been used extensively for the study of phloem contents. Alternative sampling techniques are spontaneous exudation upon wounding that only works in a few plant species, and the popular EDTA-facilitated exudation technique. These approaches have allowed fundamental advances on the understanding of phloem sap composition and sieve tube physiology, which are surveyed in this review. A more complete picture of metabolites, ions, proteins and RNAs present in phloem sap is now available, which has provided large evidence for the phloem role as a signalling network in addition to its primary role in partitioning of photo-assimilates. Thus, phloem sap sampling methods can have remarkable applications to analyse plant nutrition, physiology and defence responses. Since aphid behaviour is suspected to be affected by phloem sap quality, attempts to manipulate phloem sap content were recently undertaken based on deregulation in mutant plants of genes controlling amino acid or sugar content of phloem sap. This opens up new strategies to control aphid settlement on a plant host

Above–belowground herbivore interactions in mixed plant communities are influenced by altered precipitation patterns

Root- and shoot-feeding herbivores have the capacity to influence one another by modifying the chemistry of the shared host plant. This can alter rates of nutrient mineralization and uptake by neighboring plants and influence plant–plant competition, particularly in mixtures combining grasses and legumes. Root herbivory-induced exudation of nitrogen (N) from legume roots, for example, may increase N acquisition by co-occurring grasses, with knock-on effects on grassland community composition. Little is known about how climate change may affect these interactions, but an important and timely question is how will grass–legume mixtures respond in a future with an increasing reliance on legume N mineralization in terrestrial ecosystems. Using a model grass–legume mixture, this study investigated how simultaneous attack on lucerne (Medicago sativa) by belowground weevils (Sitona discoideus) and aboveground aphids (Acyrthosiphon pisum) affected a neighboring grass (Phalaris aquatica) when subjected to drought, ambient, and elevated precipitation. Feeding on rhizobial nodules by weevil larvae enhanced soil water retention under ambient and elevated precipitation, but only when aphids were absent. While drought decreased nodulation and root N content in lucerne, grass root and shoot chemistry were unaffected by changes in precipitation. However, plant communities containing weevils but not aphids showed increased grass height and N concentrations, most likely associated with the transfer of N from weevil-attacked lucerne plants containing more nodules and higher root N concentrations compared with insect-free plants. Drought decreased aphid abundance by 54% but increased total and some specific amino acid concentrations (glycine, lysine, methionine, tyrosine, cysteine, histidine, arginine, aspartate, and glutamate), suggesting that aphid declines were being driven by other facets of drought (e.g., reduced phloem hydraulics). The presence of weevil larvae belowground decreased aphid numbers by 30%, likely associated with a significant reduction in proline in weevil-treated lucerne plants. This study demonstrates how predicted changes to precipitation patterns and indirect interactions between herbivores can alter the outcome of competition between N-fixing legumes and non-N-fixing grasses, with important implications for plant community structure and productivity

Plant immunity in plant-aphid interactions

Aphids are economically important pests that cause extensive feeding damage and transmit viruses. While some species have a broad host range and cause damage to a variety of crops, others are restricted to only closely related plant species. While probing and feeding aphids secrete saliva, containing effectors, into their hosts to manipulate host cell processes and promote infestation. Aphid effector discovery studies pointed out parallels between infection and infestation strategies of plant pathogens and aphids. Interestingly, resistance to some aphid species is known to involve plant resistance proteins with a typical NB-LRR domain structure. Whether these resistance proteins indeed recognize aphid effectors to trigger ETI remains to be elucidated. In addition, it was recently shown that unknown aphid derived elicitors can initiate ROS (reactive oxygen species) production and callose deposition and that these responses were dependent on BAK1 (BRASSINOSTERIOD INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1) which is a key component of the plant immune system. In addition, BAK-1 contributes to nonhost resistance to aphids pointing to another parallel between plant-pathogen and –aphid interactions. Understanding the role of plant immunity and non-host resistance to aphids is essential to generate durable and sustainable aphid control strategies. Although insect behavior plays a role in host selection and non-host resistance, an important observation is that aphids interact with nonhost plants by probing the leaf surface, but are unable to feed or establish colonization. Therefore, we hypothesize that aphids interact with nonhost plants at the molecular level, but are potentially not successful in suppressing plant defenses and/or releasing nutrients

Lyplal1 is dispensable for normal fat deposition in mice.

Genome-wide association studies (GWAS) have detected association between variants in or near the Lysophospholipase-like 1 (LYPLAL1) locus and metabolic traits, including central obesity, fatty liver and waist-to-hip ratio. LYPLAL1 is also known to be upregulated in the adipose tissue of obese patients. However, the physiological role of LYPLAL1 is not understood. To investigate the function of Lyplal1 in vivo we investigated the phenotype of the Lyplal1tm1a(KOMP)Wtsi homozygous mouse. Body composition was unaltered in Lyplal1 knockout mice as assessed by dual-energy X-ray absorptiometry (DEXA) scanning, both on normal chow and on a high-fat diet. Adipose tissue distribution between visceral and subcutaneous fat depots was unaltered, with no change in adipocyte cell size. The response to both insulin and glucose dosing was normal in Lyplal1tm1a(KOMP)Wtsi homozygous mice, with normal fasting blood glucose concentrations. RNAseq analysis of liver, muscle and adipose tissue confirmed that Lyplal1 expression was ablated with minimal additional changes in gene expression. These results suggest that Lyplal1 is dispensable for normal mouse metabolic physiology and that despite having been maintained through evolution Lyplal1 is not an essential gene, suggesting possible functional redundancy. Further studies will be required to clarify its physiological role

In planta function of compatible solute transporters of the AtProT family

The three proline transporters of Arabidopsis thaliana (AtProTs) transport the compatible solutes proline and glycine betaine and the stress-induced compound γ-aminobutyric acid when expressed in heterologous systems. The aim of the present study was to show transport and physiological relevance of these three AtProTs in planta. Using single, double, and triple knockout mutants and AtProT-overexpressing lines, proline content, growth on proline, transport of radiolabelled betaine, and expression of AtProT genes and enzymes of proline metabolism were analysed. AtProT2 was shown to facilitate uptake of L- and D-proline as well as [14C]glycine betaine in planta, indicating a role in the import of compatible solutes into the root. Toxic concentrations of L- and D-proline resulted in a drastic growth retardation of AtProT-overexpressing plants, demonstrating the need for a precise regulation of proline uptake and/or distribution. Furthermore evidence is provided that AtProT genes are highly expressed in tissues with elevated proline content—that is, pollen and leaf epidermis

Adaptation of aphid stylectomy for analyses of proteins and mRNAs in barley phloem sap

Sieve tubes are transport conduits not only for photoassimilates but also for macromolecules and other compounds that are involved in sieve tube maintenance and systemic signalling. In order to gain sufficient amounts of pure phloem exudates from barley plants for analyses of the protein and mRNA composition, a previously described stylectomy set-up was optimized. Aphids were placed in sealed cages, which, immediately after microcauterization of the stylets, were flooded with water-saturated silicon oil. The exuding phloem sap was collected with a capillary connected to a pump. Using up to 30 plants and 600 aphids (Rhopalosiphum padi) in parallel, an average of 10 μl of phloem sap could be obtained within 6 h of sampling. In first analyses of the macromolecular content, eight so far unknown phloem mRNAs were identified by cDNA-amplified fragment length polymorphism. Transcripts in barley phloem exudates are related to metabolism, signalling, and pathogen defence, for example coding for a protein kinase and a pathogen- and insect-responsive WIR1A (wheat-induced resistance 1A)-like protein. Further, one-dimensional gel electrophoresis and subsequent partial sequencing by mass spectrometry led to the identification of seven major proteins with putative functions in stress responses and transport of mRNAs, proteins, and sugars. Two of the discovered proteins probably represent isoforms of a new phloem-mobile sucrose transporter. Notably, two-dimensional electrophoresis confirmed that there are >250 phloem proteins awaiting identification in future studies

Further Support to the Uncoupling-to-Survive Theory: The Genetic Variation of Human UCP Genes Is Associated with Longevity

In humans Uncoupling Proteins (UCPs) are a group of five mitochondrial inner membrane transporters with variable tissue expression, which seem to function as regulators of energy homeostasis and antioxidants. In particular, these proteins uncouple respiration from ATP production, allowing stored energy to be released as heat. Data from experimental models have previously suggested that UCPs may play an important role on aging rate and lifespan. We analyzed the genetic variability of human UCPs in cohorts of subjects ranging between 64 and 105 years of age (for a total of 598 subjects), to determine whether specific UCP variability affects human longevity. Indeed, we found that the genetic variability of UCP2, UCP3 and UCP4 do affect the individual's chances of surviving up to a very old age. This confirms the importance of energy storage, energy use and modulation of ROS production in the aging process. In addition, given the different localization of these UCPs (UCP2 is expressed in various tissues including brain, hearth and adipose tissue, while UCP3 is expressed in muscles and Brown Adipose Tissue and UCP4 is expressed in neuronal cells), our results may suggest that the uncoupling process plays an important role in modulating aging especially in muscular and nervous tissues, which are indeed very responsive to metabolic alterations and are very important in estimating health status and survival in the elderly