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International audience; Over the past few decades, various techniques have been developed and optimized for the accurate measurement of RNA abundance in cells or tissues. These methods have been instrumental in gaining insight in complex systems such as host-symbiont associations. The pea aphid model has recently emerged as a powerful and experimentally tractable system for studying symbiotic relationships and it is the subject of a growing number of molecular studies. Nevertheless, the lack of standardized protocols for the collection of bacteriocytes, the specialized host cells harboring the symbionts, has limited its use. This chapter provides a simple, step-by-step dissection protocol for the rapid isolation of aphid bacteriocytes. We then describe an adapted protocol for efficient extraction and purification of bacteriocyte RNA that can be used for most downstream transcriptomic analyses.

Additional file 4:List of the B. aphidicola TUs predicted with DisTer. (PDF 132 KB)

Additional file 12:Distribution of E. coli TUs in the Buchnera TU classes. (PDF 89 KB)

Additional file 11:Buchnera gene Ka distributions. (PDF 131 KB)

Additional file 1:Definition of the adjacent gene-pairs types. (PDF 82 KB)

Additional file 8:Buchnera vs. E. coli gene pair status comparison. (PDF 91 KB)

Additional file 3:Quality of the predictions for the three predictor models of DisTer. (PDF 126 KB)

Additional file of Structure and dynamics of the operon map of Buchnera aphidicola sp. strain APS

Additional file 7:Description of the Buchnera TU types defined by comparison with homologous E. coli TUs. (PDF 147 KB)

Additional file 2:TU predictor models evaluated during DisTer construction. (PDF 119 KB)

Additional file 9:Intergenic distance characteristics for each pair type. (PDF 87 KB)

Additional file 13:Counts of ancestral genes conserved or not conserved in Buchnera depending on their regulation in E. coli. (PDF 87 KB)

Additional file 5:Pairs of adjacent genes for which the STU status was experimentally validated by RT-PCR. (PDF 97 KB)

Additional file 10:Distributions of the scores of the predicted σ70 promoters. (PDF 124 KB)

Additional file 6:Experimental validation by RT-PCR for 4 complete operons. (PDF 260 KB)

Symbiotic associations play a pivotal role in multicellular life by facilitating acquisition of new traits and expanding the ecological capabilities of organisms. In insects that are obligatorily dependent on intracellular bacterial symbionts, novel host cells (bacteriocytes) or organs (bacteriomes) have evolved for harboring beneficial microbial partners. The processes regulating the cellular life cycle of these endosymbiont-bearing cells, such as the cell-death mechanisms controlling their fate and elimination in response to host physiology, are fundamental questions in the biology of symbiosis. Here we report the discovery of a cell-death process involved in the degeneration of bacteriocytes in the hemipteran insectThis process is activated progressively throughout aphid adulthood and exhibits morphological features distinct from known cell-death pathways. By combining electron microscopy, immunohistochemistry, and molecular analyses, we demonstrated that the initial event of bacteriocyte cell death is the cytoplasmic accumulation of nonautophagic vacuoles, followed by a sequence of cellular stress responses including the formation of autophagosomes in intervacuolar spaces, activation of reactive oxygen species, andendosymbiont degradation by the lysosomal system. We showed that this multistep cell-death process originates from the endoplasmic reticulum, an organelle exhibiting a unique reticular network organization spread throughout the entire cytoplasm and surroundingendosymbionts. Our findings provide insights into the cellular and molecular processes that coordinate eukaryotic host and endosymbiont homeostasis and death in a symbiotic system and shed light on previously unknown aspects of bacteriocyte biological functioning. ispartof: Proceedings of the National Academy of Sciences of the United States of America vol:115 issue:8 pages:E1819-E1828 ispartof: location:United States status: published

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Aphids, important agricultural pests, can grow and reproduce thanks to their intimate symbiosis with the gamma-proteobacterium Buchnera aphidicola that furnishes them with essential amino acids lacking in their phloem sap diet. To study how B. aphidicola, with its reduced genome containing very few transcriptional regulators, responds to variations in the metabolic requirements of its host, we concentrated on the leucine metabolic pathway. We show that leucine is a limiting factor for aphid growth and it displays a stimulatory feeding effect. Our metabolic analyses demonstrate that symbiotic aphids are able to respond to leucine starvation or excess by modulating the neosynthesis of this amino acid. At a molecular level, this response involves an early important transcriptional regulation (after 12 h of treatment) followed by a moderate change in the pLeu plasmid copy number. Both responses are no longer apparent after 7 days of treatment. These experimental data are discussed in the light of a re-annotation of the pLeu plasmid regulatory elements. Taken together, our data show that the response of B. aphidicola to the leucine demand of its host is multimodal and dynamically regulated, providing new insights concerning the genetic regulation capabilities of this bacterium in relation to its symbiotic functions.

Pea albumin 1b (PA1b), a plant entomotoxin isolated from Pisum sativum (Delobel et al., 1998), was used to generate transgenic rice lines resistant to the stored-product insect pest Sitophilus oryzae. PA1b is the result of the post-transcriptional cleavage of the albumin polyprotein PA1 which also leads to the release of a second peptide, PA1a (53 amino acids). PA1b is a cysteine-rich peptide of 37 amino acids belonging to the cystine-knot family (Jouvensal et al., 2003). In pea, different nucleotidic sequences (Louis et al., 2004) and protein extracts (Taylor et al., 2004) corresponding to PA1b have been isolated underlying the multigenic character of this new family of plant entomotoxin. Two isoforms of pa1 gene (AJ574794 and AJ574795), placed under the control of the maize ubiquitin promoter, were introduced in rice (cv. Zhong Zuo 321) by Agrobacterium tumefaciens-mediated transformation (Sallaud et al., 2003) and studied separately. 24 T0 events of each isoform were regenerated and characterized by RT-PCR and Southern blot. T1 and T2 seeds were analyzed by Near-Infrared Spectroscopy (NIRS) and by in situ western hybridization (Qu et al., 2003). The latter technique allowed to localize PA1b accumulation in embryo and aleurone layer. Resistance of T2 elite lines was tested in bioassay by feeding 20 rice weevil adults with 5 transgenic seeds. Maximum of mortality was raised by line 30.16 after 5 days (AJ574795) and by line 55.19 after 4 days (AJ574794) of bioassay. Resistance of T3 seeds will be examined in selected homozygous T2 lines.