Co-option of a conserved host glutamine transporter facilitates aphid/Buchnera metabolic integration.

Organisms across the tree of life colonize novel environments by partnering with bacterial symbionts. These symbioses are characterized by intimate integration of host/endosymbiont biology at multiple levels, including metabolically. Metabolic integration is particularly important for sap-feeding insects and their symbionts, which supplement nutritionally unbalanced host diets. Many studies reveal parallel evolution of host/endosymbiont metabolic complementarity in amino acid biosynthesis, raising questions about how amino acid metabolism is regulated, how regulatory mechanisms evolve, and the extent to which similar mechanisms evolve in different systems. In the aphid/Buchnera symbiosis, the transporter ApGLNT1 (Acyrthosiphon pisum glutamine transporter 1) supplies glutamine, an amino donor in transamination reactions, to bacteriocytes (where Buchnera reside) and is competitively inhibited by Buchnera-supplied arginine-consistent with a role regulating amino acid metabolism given host demand for Buchnera-produced amino acids. We examined how ApGLNT1 evolved a regulatory role by functionally characterizing orthologs in insects with and without endosymbionts. ApGLNT1 orthologs are functionally similar, and orthology searches coupled with homology modeling revealed that GLNT1 is ancient and structurally conserved across insects. Our results indicate that the ApGLNT1 symbiotic regulatory role is derived from its ancestral role and, in aphids, is likely facilitated by loss of arginine biosynthesis through the urea cycle. Given consistent loss of host arginine biosynthesis and retention of endosymbiont arginine supply, we hypothesize that GLNT1 is a general mechanism regulating amino acid metabolism in sap-feeding insects. This work fills a gap, highlighting the broad importance of co-option of ancestral proteins to novel contexts in the evolution of host/symbiont systems.

Potato leafroll virus reduces Buchnera aphidocola titer and alters vector transcriptome responses.

Viruses in the Luteoviridae family, such as Potato leafroll virus (PLRV), are transmitted by aphids in a circulative and nonpropagative mode. This means the virions enter the aphid body through the gut when they feed from infected plants and then the virions circulate through the hemolymph to enter the salivary glands before being released into the saliva. Although these viruses do not replicate in their insect vectors, previous studies have demonstrated viruliferous aphid behavior is altered and the obligate symbiont of aphids, Buchnera aphidocola, may be involved in transmission. Here we provide the transcriptome of green peach aphids (Myzus persicae) carrying PLRV and virus-free control aphids using Illumina sequencing. Over 150 million paired-end reads were obtained through Illumina sequencing, with an average of 19 million reads per library. The comparative analysis identified 134 differentially expressed genes (DEGs) between the M. persicae transcriptomes, including 64 and 70 genes that were up- and down-regulated in aphids carrying PLRV, respectively. Using functional classification in the GO databases, 80 of the DEGs were assigned to 391 functional subcategories at category level 2. The most highly up-regulated genes in aphids carrying PLRV were cytochrome p450s, genes related to cuticle production, and genes related to development, while genes related to heat shock proteins, histones, and histone modification were the most down-regulated. PLRV aphids had reduced Buchnera titer and lower abundance of several Buchnera transcripts related to stress responses and metabolism. These results suggest carrying PLRV may reduce both aphid and Buchnera genes in response to stress. This work provides valuable basis for further investigation into the complicated mechanisms of circulative and nonpropagative transmission.

B-vitamin nutrition in the pea aphid-Buchnera symbiosis.

Various insects that utilize vitamin-deficient diets derive a supplementary supply of these micronutrients from their symbiotic microorganisms. Here, we tested the inference from genome annotation that the symbiotic bacterium Buchnera aphidicola in the pea aphid Acyrthosiphon pisum provides the insect with vitamins B2 and B5 but no other B-vitamins. Contrary to expectation, aphid survival over five days of larval development on artificial diets individually lacking each B-vitamin not synthesized by Buchnera was not significantly reduced, despite significantly lower carcass B1, B3, B6 and B7 concentrations in the aphids on diets lacking each of these B-vitamins than on the vitamin-complete diet. Aphid survival was, however, significantly reduced on diet containing low concentrations (≤0.2 mM) or no pantothenate (B5). Complementary transcriptome analysis revealed low abundance of the sense-transcript, but high abundance of the antisense transcript, of the Buchnera gene panC encoding the enzyme mediating the terminal reaction in pantothenate synthesis. We hypothesize that metabolic constraints or antisense transcripts may reduce Buchnera-mediated production of pantothenate, resulting in poor aphid performance on pantothenate-free diets. The discrepancy between predictions from genome data and empirical data illustrates the need for physiological study to test functional inferences made from genome annotations.

GroEL from the endosymbiont Buchnera aphidicola betrays the aphid by triggering plant defense.

Aphids are sap-feeding plant pests and harbor the endosymbiont Buchnera aphidicola, which is essential for their fecundity and survival. During plant penetration and feeding, aphids secrete saliva that contains proteins predicted to alter plant defenses and metabolism. Plants recognize microbe-associated molecular patterns and induce pattern-triggered immunity (PTI). No aphid-associated molecular pattern has yet been identified. By mass spectrometry, we identified in saliva from potato aphids (Macrosiphum euphorbiae) 105 proteins, some of which originated from Buchnera, including the chaperonin GroEL. Because GroEL is a widely conserved bacterial protein with an essential function, we tested its role in PTI. Applying or infiltrating GroEL onto Arabidopsis (Arabidopsis thaliana) leaves induced oxidative burst and expression of PTI early marker genes. These GroEL-induced defense responses required the known coreceptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1. In addition, in transgenic Arabidopsis plants, inducible expression of groEL activated PTI marker gene expression. Moreover, Arabidopsis plants expressing groEL displayed reduced fecundity of the green peach aphid (Myzus persicae), indicating enhanced resistance against aphids. Furthermore, delivery of GroEL into tomato (Solanum lycopersicum) or Arabidopsis through Pseudomonas fluorescens, engineered to express the type III secretion system, also reduced potato aphid and green peach aphid fecundity, respectively. Collectively our data indicate that GroEL is a molecular pattern that triggers PTI.

Aphid genome expression reveals host-symbiont cooperation in the production of amino acids.

The evolution of intimate symbiosis requires the coordination of gene expression and content between the distinct partner genomes; this coordination allows the fusion of capabilities of each organism into a single integrated metabolism. In aphids, the 10 essential amino acids are scarce in the phloem sap diet and are supplied by the obligate bacterial endosymbiont (Buchnera), which lives inside specialized cells called bacteriocytes. Although Buchnera's genome encodes most genes for essential amino acid biosynthesis, several genes in essential amino acid pathways are missing, as are most genes for production of nonessential amino acids. Additionally, it is unresolved whether the supply of nitrogen for amino acid biosynthesis is supplemented by recycling of waste ammonia. We compared pea aphid gene expression between bacteriocytes and other body tissues using RNA sequencing and pathway analysis and exploiting the genome sequences available for both partners. We found that 26 genes underlying amino acid biosynthesis were up-regulated in bacteriocytes. Seven of these up-regulated genes fill the gaps of Buchnera's essential amino acid pathways. In addition, genes underlying five nonessential amino acid pathways lost from Buchnera are up-regulated in bacteriocytes. Finally, our results reveal that two genes, glutamine synthetase and glutamate synthase, which potentially work together in the incorporation of ammonium nitrogen into glutamate (GOGAT) cycle to assimilate ammonia into glutamate, are up-regulated in bacteriocytes. Thus, host gene expression and symbiont capabilities are closely integrated within bacteriocytes, which function as specialized organs of amino acid production. Furthermore, the GOGAT cycle may be a key source of nitrogen fueling the integrated amino acid metabolism of the aphid-Buchnera partnership.

The impact of host plant on the abundance and function of symbiotic bacteria in an aphid.

The black-bean aphid Aphis fabae bears populations of coccoid symbiotic bacteria Buchnera spp. at 2.0-3.2 x 10(7)cells mg(-1)aphid mass and rod-shaped secondary symbionts of uncertain taxonomic affiliation at 0.1-0.6 x 10(7)cells mg(-1)aphid mass. Buchnera provides essential amino acids, supplementing the poor supply in the aphid diet of plant phloem sap. Comparison of the performance of A. fabae containing and experimentally deprived of their bacteria showed that the bacteria caused increased larval mass of aphids reared on Chenopodium album and Papaver dubium plants, but not when reared on Lamium purpureum. In the aphids reared on L. purpureum, the density of the bacteria, especially the secondary symbionts, was significantly elevated, and bacterial-mediated production of the essential amino acid threonine was reduced, even though the essential amino acid content of phloem exudates from L. purpureum had a low threonine content. It is proposed that the shortfall in threonine, possibly compounded by the high density of secondary symbionts, may contribute to the poor performance of the aphids on L. purpureum. This study offers the first evidence to suggest plant-mediated interference with the nutritional function of symbiotic bacteria in any phytophagous insect.

Expression of host S-adenosylmethionine decarboxylase gene and polyamine composition in aphid bacteriocytes.

Differential cDNA display and quantitative RT-PCR revealed that mRNA of host S-adenosylmethionine decarboxylase (SAMDC) was abundant only in the aphid endosymbiotic system well organized in young hosts, suggesting that SAMDC plays some important roles in the system. SAMDC is a key enzyme to synthesize polyamines that are known to be involved in a large array of biological events including protein synthesis, DNA stabilization, DNA replication, and cell proliferation. As the first step to investigate roles of polyamines in the endosymbiotic system, polyamine composition in bacteriocytes was determined by high performance liquid chromatography. As a result, we found that bacteriocytes contained virtually an only single polyamine, spermidine. The spermidine content of bacteriocytes fluctuated with time in the course of development and aging of the host aphid. This is the first report of polyamine assessment in a prokaryote-eukaryote endocellular symbiotic system, which demonstrated a unique polyamine composition.

Identifying the determinants in the equatorial domain of Buchnera GroEL implicated in binding Potato leafroll virus.

Luteoviruses avoid degradation in the hemolymph of their aphid vector by interacting with a GroEL homolog from the aphid's primary endosymbiotic bacterium (Buchnera sp.). Mutational analysis of GroEL from the primary endosymbiont of Myzus persicae (MpB GroEL) revealed that the amino acids mediating binding of Potato leafroll virus (PLRV; Luteoviridae) are located within residues 9 to 19 and 427 to 457 of the N-terminal and C-terminal regions, respectively, of the discontinuous equatorial domain. Virus overlay assays with a series of overlapping synthetic decameric peptides and their derivatives demonstrated that R13, K15, L17, and R18 of the N-terminal region and R441 and R445 of the C-terminal region of the equatorial domain of GroEL are critical for PLRV binding. Replacement of R441 and R445 by alanine in full-length MpB GroEL and in MpB GroEL deletion mutants reduced but did not abolish PLRV binding. Alanine substitution of either R13 or K15 eliminated the PLRV-binding capacity of the other and those of L17 and R18. In the predicted tertiary structure of GroEL, the determinants mediating virus binding are juxtaposed in the equatorial plain.