Daniel R.G. Price

RNAi-mediated crop protection against insects

Downregulation of the expression of specific genes through RNA interference (RNAi), has been widely used for genetic research in insects. The method has relied on the injection of double-stranded RNA (dsRNA), which is not possible for practical applications in crop protection. By contrast, specific suppression of gene expression in nematodes is possible through feeding with dsRNA. This approach was thought to be unfeasible in insects, but recent results have shown that dsRNA fed as a diet component can be effective in downregulating targeted genes. More significantly, expression of dsRNA directed against suitable insect target genes in transgenic plants has been shown to give protection against pests, opening the way for a new generation of insect-resistant crops.

Aphid amino acid transporter regulates glutamine supply to intracellular bacterial symbionts

Significance Nutritional bacterial endosymbionts are housed in specialized host cells and are partitioned from the host cell cytoplasm by a host-derived symbiosomal membrane. This cellular organization isolates bacterial symbionts from nutrient pools in the host cell and makes possible host control of nutrient supply to bacterial symbionts. Here, using the aphid–Buchnera nutritional endosymbiosis, we demonstrate that the most active host glutamine (precursor) transporter, Acyrthosiphon pisum glutamine transporter 1, is competitively inhibited by arginine (a Buchnera-synthesized end product). We propose a model of endosymbiosis regulation in which precursor transport is regulated by a symbiont-synthesized end product. Thus, we provide insights into the molecular mechanism of host control of bacterial endosymbiont essential nutrient biosynthesis. Endosymbiotic associations have played a major role in evolution. However, the molecular basis for the biochemical interdependence of these associations remains poorly understood. The aphid–Buchnera endosymbiosis provides a powerful system to elucidate how these symbioses are regulated. In aphids, the supply of essential amino acids depends on an ancient nutritional symbiotic association with the gamma-proteobacterium Buchnera aphidicola. Buchnera cells are densely packed in specialized aphid bacteriocyte cells. Here we confirm that five putative amino acid transporters are highly expressed and/or highly enriched in Acyrthosiphon pisum bacteriocyte tissues. When expressed in Xenopus laevis oocytes, two bacteriocyte amino acid transporters displayed significant levels of glutamine uptake, with transporter ACYPI001018, LOC100159667 (named here as Acyrthosiphon pisum glutamine transporter 1, ApGLNT1) functioning as the most active glutamine transporter. Transporter ApGLNT1 has narrow substrate selectivity, with high glutamine and low arginine transport capacity. Notably, ApGLNT1 has high binding affinity for arginine, and arginine acts as a competitive inhibitor for glutamine transport. Using immunocytochemistry, we show that ApGLNT1 is localized predominantly to the bacteriocyte plasma membrane, a location consistent with the transport of glutamine from A. pisum hemolymph to the bacteriocyte cytoplasm. On the basis of functional transport data and localization, we propose a substrate feedback inhibition model in which the accumulation of the essential amino acid arginine in A. pisum hemolymph reduces the transport of the precursor glutamine into bacteriocytes, thereby regulating amino acid biosynthesis in the bacteriocyte. Structural similarities in the arrangement of hosts and symbionts across endosymbiotic systems suggest that substrate feedback inhibition may be mechanistically important in other endosymbioses.

Sweet problems: insect traits defining the limits to dietary sugar utilisation by the pea aphid, Acyrthosiphon pisum.

SUMMARY Plant phloem sap is an extreme diet for animals, partly because of its high and variable sugar content. The physiological and feeding traits of the pea aphid Acyrthosiphon pisum that define the upper and lower limits to the range of dietary sucrose concentrations utilised by this insect were determined principally using chemically defined diets containing 0.125–1.5 mol l–1 sucrose. On the diets with 0.125 mol l–1 and 1.5 mol l–1 sucrose, the aphids died as larvae within 8 and 14 days of birth, respectively. On the other diets, 60–96% of aphids developed to adulthood, and the 0.5 mol l–1 and 0.75 mol l–1 diets supported the highest fecundity. The diet with 0.125 mol l–1 sucrose was ingested at 36% of the rate of the 0.25 mol l–1 sucrose diet, but >90% of ingested sucrose-carbon was assimilated on both diets. This suggests that the lower limit is dictated by the aphid feeding response, specifically, a requirement for a minimal concentration of sucrose for sustained feeding. The haemolymph osmotic pressure of aphids on diets with 0.125–1.5 mol l–1 sucrose was up to 68% higher than on 0.125–1.0 mol l–1 sucrose diets, but diet consumption and sucrose-carbon assimilation was not reduced on the very high sucrose diets relative to 1.0 mol l–1 sucrose. This suggests that failure of the osmoregulatory capacity of the insects on high sucrose diets may define the upper limit to the range of dietary sucrose utilised by the aphids. The mean haemolymph osmotic pressure of aphids on plants with phloem sap containing 0.37–0.97 mol l–1 sucrose was 1.61±0.063 MPa (mean ± s.e.m.), not significantly different from that (1.47±0.059 MPa) on diets with 0.25–1.0 mol l–1 sucrose. It is concluded that the osmoregulatory response of aphids to diets and plants are comparable, and, more generally, that the feeding and osmoregulatory capabilities of the aphids are compatible with the phloem sugar levels commonly encountered by aphids feeding on plants.

A venom metalloproteinase from the parasitic wasp Eulophus pennicornis is toxic towards its host, tomato moth (Lacanobia oleracae)

Three genes encoding clan MB metalloproteinases (EpMP1–3) were identified from venom glands of the ectoparasitic wasp Eulophus pennicornis. The derived amino acid sequences predict mature proteins of approximately 46 kDa, with a novel two‐domain structure comprising a C‐terminal reprolysin domain, and an N‐terminal domain of unknown function. EpMP3 expressed as a recombinant protein in Pichia pastoris had gelatinase activity, which was inhibited by EDTA. Injection of recombinant EpMP3 into fifth instar Lacanobia oleracea (host) larvae resulted in partial insect mortality associated with the moult to sixth instar, with surviving insects showing retarded development and growth. EpMP3 is expressed specifically in venom glands. These results suggest that EpMP3 is a functional component of Eulophus venom, which is able to manipulate host development.

Genome Expansion and Differential Expression of Amino Acid Transporters at the Aphid/Buchnera Symbiotic Interface

In insects, some of the most ecologically important symbioses are nutritional symbioses that provide hosts with novel traits and thereby facilitate exploitation of otherwise inaccessible niches. One such symbiosis is the ancient obligate intracellular symbiosis of aphids with the γ-proteobacteria, Buchnera aphidicola. Although the nutritional basis of the aphid/Buchnera symbiosis is well understood, the processes and structures that mediate the intimate interactions of symbiotic partners remain uncharacterized. Here, using a de novo approach, we characterize the complement of 40 amino acid polyamine organocation (APC) superfamily member amino acid transporters (AATs) encoded in the genome of the pea aphid, Acyrthosiphon pisum. We find that the A. pisum APC superfamily is characterized by extensive gene duplications such that A. pisum has more APC superfamily transporters than other fully sequenced insects, including a ten paralog aphid-specific expansion of the APC transporter slimfast. Detailed expression analysis of 17 transporters selected on the basis of their phylogenetic relationship to five AATs identified in an earlier bacteriocyte expressed sequence tag study distinguished a subset of eight transporters that have been recruited for amino acid transport in bacteriocyte cells at the symbiotic interface. These eight transporters include transporters that are highly expressed and/or highly enriched in bacteriocytes and intriguingly, the four AATs that show bacteriocyte-enriched expression are all members of gene family expansions, whereas three of the four that are highly expressed but not enriched in bacteriocytes retain one-to-one orthology with transporters in other genomes. Finally, analysis of evolutionary rates within the large A. pisum slimfast expansion demonstrated increased rates of molecular evolution coinciding with two major shifts in expression: 1) a loss of gut expression and possibly a gain of bacteriocyte expression and 2) loss of expression in all surveyed tissues in asexual females. Taken together, our characterization of nutrient AATs at the aphid/Buchnera symbiotic interface provides the first examination of the processes and structures operating at the interface of an obligate intracellular insect nutritional symbiosis, offering unique insight into the types of genomic change that likely facilitated evolutionary maintenance of the symbiosis.

Rapid transcriptional plasticity of duplicated gene clusters enables a clonally reproducing aphid to colonise diverse plant species

BackgroundThe prevailing paradigm of host-parasite evolution is that arms races lead to increasing specialisation via genetic adaptation. Insect herbivores are no exception and the majority have evolved to colonise a small number of closely related host species. Remarkably, the green peach aphid, Myzus persicae, colonises plant species across 40 families and single M. persicae clonal lineages can colonise distantly related plants. This remarkable ability makes M. persicae a highly destructive pest of many important crop species.ResultsTo investigate the exceptional phenotypic plasticity of M. persicae, we sequenced the M. persicae genome and assessed how one clonal lineage responds to host plant species of different families. We show that genetically identical individuals are able to colonise distantly related host species through the differential regulation of genes belonging to aphid-expanded gene families. Multigene clusters collectively upregulate in single aphids within two days upon host switch. Furthermore, we demonstrate the functional significance of this rapid transcriptional change using RNA interference (RNAi)-mediated knock-down of genes belonging to the cathepsin B gene family. Knock-down of cathepsin B genes reduced aphid fitness, but only on the host that induced upregulation of these genes.ConclusionsPrevious research has focused on the role of genetic adaptation of parasites to their hosts. Here we show that the generalist aphid pest M. persicae is able to colonise diverse host plant species in the absence of genetic specialisation. This is achieved through rapid transcriptional plasticity of genes that have duplicated during aphid evolution.

Sugar transporters of the major facilitator superfamily in aphids; from gene prediction to functional characterization.

Analysis of the pea aphid (Acyrthosiphon pisum) genome using signatures specific to the Major Facilitator Superfamily (Pfam Clan CL0015) and the Sugar_tr family (Pfam Family PF00083) has identified 54 genes encoding potential sugar transporters, of which 38 have corresponding ESTs. Twenty‐nine genes contain the InterPro IPR003663 hexose transporter signature. The protein encoded by Ap_ST3, the most abundantly expressed sugar transporter gene, was functionally characterized by expression as a recombinant protein. Ap_ST3 acts as a low‐affinity uniporter for fructose and glucose that does not depend on Na+ or H+ for activity. Ap_ST3 was expressed at elevated levels in distal gut tissue, consistent with a role in gut sugar transport. The A. pisum genome shows evidence of duplications of sugar transporter genes.

A new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) affects Soybean Asian rust (Phakopsora pachyrhizi) spore germination

BackgroundAsian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the pGAPZα-B vector for expression in Pichia pastoris.ResultsA cDNA encoding a chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP), was isolated from leaves. The amino acid sequence predicts a (β/α)8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins; GH18), and shares similarity with other GH18 members, although it lacks the glutamic acid residue essential for catalysis, which is replaced by glutamine. CaclXIP was expressed as a recombinant protein in Pichia pastoris. Enzymatic assay showed that purified recombinant CaclXIP had only residual chitinolytic activity. However, it inhibited xylanases from Acrophialophora nainiana by approx. 60% when present at 12:1 (w/w) enzyme:inhibitor ratio. Additionally, CaclXIP at 1.5 μg/μL inhibited the germination of spores of Phakopsora pachyrhizi by 45%.ConclusionsOur data suggests that CaclXIP belongs to a class of naturally inactive chitinases that have evolved to act in plant cell defence as xylanase inhibitors. Its role on inhibiting germination of fungal spores makes it an eligible candidate gene for the control of Asian rust.

A substrate ambiguous enzyme facilitates genome reduction in an intracellular symbiont

BackgroundGenome evolution in intracellular microbial symbionts is characterized by gene loss, generating some of the smallest and most gene-poor genomes known. As a result of gene loss these genomes commonly contain metabolic pathways that are fragmented relative to their free-living relatives. The evolutionary retention of fragmented metabolic pathways in the gene-poor genomes of endosymbionts suggests that they are functional. However, it is not always clear how they maintain functionality. To date, the fragmented metabolic pathways of endosymbionts have been shown to maintain functionality through complementation by host genes, complementation by genes of another endosymbiont and complementation by genes in host genomes that have been horizontally acquired from a microbial source that is not the endosymbiont. Here, we demonstrate a fourth mechanism.ResultsWe investigate the evolutionary retention of a fragmented pathway for the essential nutrient pantothenate (vitamin B5) in the pea aphid, Acyrthosiphon pisum endosymbiosis with Buchnera aphidicola. Using quantitative analysis of gene expression we present evidence for complementation of the Buchnera pantothenate biosynthesis pathway by host genes. Further, using complementation assays in an Escherichia coli mutant we demonstrate functional replacement of a pantothenate biosynthesis enzyme, 2-dehydropantoate 2-reductase (E.C. 1.1.1.169), by an endosymbiont gene, ilvC, encoding a substrate ambiguous enzyme.ConclusionsEarlier studies have speculated that missing enzyme steps in fragmented endosymbiont metabolic pathways are completed by adaptable endosymbiont enzymes from other pathways. Here, we experimentally demonstrate completion of a fragmented endosymbiont vitamin biosynthesis pathway by recruitment of a substrate ambiguous enzyme from another pathway. In addition, this work extends host/symbiont metabolic collaboration in the aphid/Buchnera symbiosis from amino acid metabolism to include vitamin biosynthesis.

Genome-wide annotation and functional identification of aphid GLUT-like sugar transporters.

BackgroundPhloem feeding insects, such as aphids, feed almost continuously on plant phloem sap, a liquid diet that contains high concentrations of sucrose (a disaccharide comprising of glucose and fructose). To access the available carbon, aphids hydrolyze sucrose in the gut lumen and transport its constituent monosaccharides, glucose and fructose. Although sugar transport plays a critical role in aphid nutrition, the molecular basis of sugar transport in aphids, and more generally across all insects, remains poorly characterized. Here, using the latest release of the pea aphid, Acyrthosiphon pisum, genome we provide an updated gene annotation and expression profile of putative sugar transporters. Finally, gut expressed sugar transporters are functionally expressed in yeast and screened for glucose and fructose transport activity.ResultsIn this study, using a de novo approach, we identified 19 sugar porter (SP) family transporters in the A. pisum genome. Gene expression analysis, based on 214, 834 A. pisum expressed sequence tags, supports 17 sugar porter family transporters being actively expressed in adult female aphids. Further analysis, using quantitative PCR identifies 4 transporters, A. pisum sugar transporter 1, 3, 4 and 9 (ApST1, ApST3, ApST4 and ApST9) as highly expressed and/or enriched in gut tissue. When expressed in a Saccharomyces cerevisiae hexose transporter deletion mutant (strain EBY.VW4000), only ApST3 (previously characterized) and ApST4 (reported here) transport glucose and fructose resulting in functional rescue of the yeast mutant. Here we characterize ApST4, a 491 amino acid protein, with 12 predicted transmembrane regions, as a facilitative glucose/fructose transporter. Finally, phylogenetic reconstruction reveals that ApST4, and related, as yet uncharacterized insect transporters are phylogenetically closely related to human GLUT (SLC2A) class I facilitative glucose/fructose transporters.ConclusionsThe gut enhanced expression of ApST4, and the transport specificity of its product is consistent with ApST4 functioning as a gut glucose/fructose transporter. Here, we hypothesize that both ApST3 (reported previously) and ApST4 (reported here) function at the gut interface to import glucose and fructose from the gut lumen.