Project description:Aphid adaptation to harsh winter conditions is illustrated by an alternation of their reproductive mode. Aphids detect photoperiod shortening by sensing the length of the night and switch from viviparous parthenogenesis in spring and summer, to oviparous sexual reproduction in autumn. The photoperiodic signal is transduced from the head to the reproductive tract to change the fate of the future oocytes from mitotic diploid embryogenesis to haploid formation of gametes. Because of viviparous parthenogenesis, the whole process takes place in three consecutive generations. To understand the molecular basis of the switch in the reproductive mode, a transcriptomic approach was used to detect significantly regulated transcripts in the heads of the pea aphid Acyrthosiphon pisum. The transcriptomic profiles of the heads of the first generation were slightly affected by photoperiod shortening. This suggests that trans-generation signaling does not occur between the grand-mothers and the viviparous embryos they contain. By analogy, many of the genes regulated in the heads of the second generation are implicated in visual functions, photoreception and cuticle structure. The modification of the cuticle could decrease the storage of N-Î²-alanyldopamine and provoke an increase in free dopamine concentration. Based in results in Drosophila, modification of the insulin pathway could cause a decrease of juvenile hormones in short-day reared aphids. Biological material for microarray experiments was prepared under two dayly photoperiodic regimes both at constant temperature of 18Â°C: i) âShort Nightâ (SN) at 16h of light and ii) âLong Nightâ (LN) at 12h of light to induce the production of sexual morphs. To initiate the experiment, two groups of 105 L3 larvae were placed either under SN or LN condition. This corresponds to generation G0. At the middle of the photophase, 25 individual were frozen when they had reached both the L4 and the wingless adult (WA) stages, in the two photoperiod conditions. The 55 remaining WA individuals (still divided in two groups) were left on 55 plants to lay their offspring: one larva of the 1st stage (L1) was kept per WA. This larva was selected among the 20 first born larvae. This is the generation G1. At the middle of the photophase, 25 individual were frozen when they had reached both the L2 and the L4 stages, in the two photoperiodic conditions. Thus, 25 individuals from 4 different stages (L4-G0, WA-G0, L2-G1 and L4-G1) were collected in the two photoperiod conditions with 3 biological replicates, forming the 24 samples used for microarray experiments. RNAs from heads of aphids from the two photoperiodic conditions were hybridized one against the other for each stage with a dye-swap.The experimental design is thus 24 arrays which corresponds to the described samples of that series.

Project description:Compatibility between aphids and plant hosts is genetically determined by both interactingorganisms. For example, plants may carry resistance (R) genes or deploy chemical defences.Aphid saliva contains many proteins that are secreted into host tissues. Subsets of these proteins are predicted to act as effectors, either subverting or triggering host immunity. However, associating particular effectors with virulence or avirulence outcomes presents challenges due to the combinatorial complexity. Here we use defined aphid and host geneticsto test for co-segregation of expressed aphid transcripts and proteins with virulent or avirulent phenotypes. We compared virulent and avirulent pea aphid parental genotypes, and their bulk segregant F1 progeny on Medicago truncatula genotypes carrying or lacking the RAP1 resistance quantitative trait locus. Differential expression analysis based on RNA sequencing of whole bodyand head samples, in combination with proteomics of saliva and salivary glands, enabled us to pinpoint proteins associated with virulence/avirulence phenotypes. There was relatively little impact of host genotype, whereas large numbers of transcripts and proteins were differentially expressed between parental aphids, likely a reflection of their classification as divergent biotypes within the pea aphid species complex. Many fewer transcripts intersected with the equivalent differential expression patterns in the bulked F1F1 progeny, providing an effective filter for removing genomic background effects. Overall, there were more upregulated genes detected in the F1 avirulent dataset compared with the virulent one. Some of the differentially expressed transcripts were also found in the differentially expressed proteomes, with aminopeptidase N proteins being the most frequent differentiallyexpressed family. In addition, a substantial proportion (23%) of salivary proteins lack annotations, suggesting that many novel functions remain to be discovered.

Project description:Compatibility between aphids and plant hosts is genetically determined by both interacting organisms. For example, plants may carry resistance (R) genes or deploy chemical defences. Aphid saliva contains many proteins that are secreted into host tissues. Subsets of these proteins are predicted to act as effectors, either subverting or triggering host immunity. However, associating particular effectors with virulence or avirulence outcomes presents challenges due to the combinatorial complexity. Here we use defined aphid and host geneticsto test for co-segregation of expressed aphid transcripts and proteins with virulent or avirulent phenotypes. We compared virulent and avirulent pea aphid parental genotypes, and their bulk segregant F1 progeny on Medicago truncatula genotypes carrying or lacking the RAP1 resistance quantitative trait locus. Differential expression analysis based on RNA sequencing of whole bodyand head samples, in combination with proteomics of saliva and salivary glands, enabled us to pinpoint proteins associated with virulence/avirulence phenotypes. There was relatively little impact of host genotype, whereas large numbers of transcripts and proteins were differentially expressed between parental aphids, likely a reflection of their classification as divergent biotypes within the pea aphid species complex. Many fewer transcripts intersected with the equivalent differential expression patterns in the bulked F1F1 progeny, providing an effective filter for removing genomic background effects. Overall, there were more upregulated genes detected in the F1 avirulent dataset compared with the virulent one. Some of the differentially expressed transcripts were also found in the differentially expressed proteomes, with aminopeptidase N proteins being the most frequent differentially expressed family. In addition, a substantial proportion (23%) of salivary proteins lack annotations, suggesting that many novel functions remain to be discovered.

Project description:Aphids, sap-sucking insects in the order Hemiptera, are among the most prolific insect vectors of plant viruses Plant viruses from the family Luteoviridae are transmitted exclusively by aphids in a circulative manner and cause significant crop yield losses. Circulative plant viruses must cross the aphid gut and other tissues prior to transmission to a new host plant. The discovery of proteins that control acquisition and transmission in the insect vector is the biggest challenge for the vector biology field and will have practical applications for growers by providing new molecular targets for the development of precision vector management tools. The green peach aphid, Myzus persicae, is a vector of the Potato leafroll virus (PLRV), a polerovirus in the Luteoviridae. PLRV transmission efficiency was significantly reduced when a clonal lineage of M. persicae was reared on turnip (T-Myzus) as compared to the weed physalis (P-Myzus). The effect on PLRV transmission efficiency was transient and caused by a host-switch response. Using 2-D DIGE, we revealed that the major difference in the proteome profile of P- and T-Myzus was the lysosomal cysteine protease cathepsin B, with multiple size and charge isoforms of this enzyme up-regulated in T-Myzus. Quantitative, shotgun proteomics revealed a specific upregulation in the expression of other lysosomal proteins in T-Myzus as compared to P-Myzus, including cathepsin B, cathepsin B-16, beta-glucuronidase, peroxidasin, legumain-like, and aminopeptidase-N. The titer of PLRV was over 1.5 fold higher in P-Myzus than in T-Myzus at 24h and 72h after the beginning of virus acquisition, suggesting that virus acquisition in P-Myzus was more efficient. Cathepsin B and PLRV localization were starkly different in P- and T-Myzus midguts, the site of PLRV acquisition into the insect. In P-Myzus midguts, an abundance of PLRV was observed inside midgut cells, and cathepsin B was sequestered in a subcellular compartment. In contrast, there is near complete co-localization of cathepsin B and PLRV at the cell membranes in viruliferous T-Myzus. Inhibition of cathepsin and other cysteine proteases with E64 restored the ability of T-Myzus to transmit PLRV in a dose-dependent manner, suggesting that the activities of lysosomal cysteine proteases at the cell membrane in T-Myzus is responsible for the change in virus transmission phenotype in these aphids. T-Myzus individuals weighed more and had more progeny than P-Myzus individuals. These data are all consistent with the hypothesis that there is an induction of lysosomal exocytos in the midgut of T-Myzus linked to the ability of the aphid to acquire PLRV. These data also show that the ability of the generalist aphid M. persicae to transmit PLRV is influenced by the host plant the aphids are reared on, information that is useful to growers for polerovirus management in field crops.

Project description:Aiming at defining the protein content and composition of a single mitochondrion of Arabidopsis thaliana, shotgun analyses were performed on purified mitochondria from cultured heterotrophic Arabidopsis cells. Based on microscopic observations on the size of mitochondria, as well as biochemical properties, such as protein concentration, average molecular mass of proteins, and the average length of the signal peptide, intensity-based absolute quantification (iBAQ) values were applied to calculate the copy number of each identified protein species within a single mitochondrion. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40,000 for Voltage-Dependent Anion Channel 1 (VDAC1) to sub-stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat (PPR) proteins that modify mitochondrial transcripts. Aiming at defining the protein content and composition of a single mitochondrion of Arabidopsis thaliana, shotgun analyses were performed on purified mitochondria from cultured heterotrophic Arabidopsis cells. Based on microscopic observations on the size of mitochondria, as well as biochemical properties, such as protein concentration, average molecular mass of proteins, and the average length of the signal peptide, intensity-based absolute quantification (iBAQ) values were applied to calculate the copy number of each identified protein species within a single mitochondrion. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40,000 for Voltage-Dependent Anion Channel 1 (VDAC1) to sub-stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat (PPR) proteins that modify mitochondrial transcripts. Aiming at defining the protein content and composition of a single mitochondrion of Arabidopsis thaliana, shotgun analyses were performed on purified mitochondria from cultured heterotrophic Arabidopsis cells. Based on microscopic observations on the size of mitochondria, as well as biochemical properties, such as protein concentration, average molecular mass of proteins, and the average length of the signal peptide, intensity-based absolute quantification (iBAQ) values were applied to calculate the copy number of each identified protein species within a single mitochondrion. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40,000 for Voltage-Dependent Anion Channel 1 (VDAC1) to sub-stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat (PPR) proteins that modify mitochondrial transcripts.

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Pea aphid parthenogenetic development

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