Proteomic analysis of secreted saliva from Russian Wheat Aphid (Diuraphis noxia Kurd.) biotypes that differ in virulence to wheat (original) (raw)
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Salivary Proteins of Russian Wheat Aphid (Hemiptera: Aphididae)
Environmental Entomology, 2010
Salivary secretions play critical roles in aphidÐ host plant interactions and are responsible for damage associated with aphid feeding. The objectives of this study were to evaluate aspects of salivation and the salivary constituents of Diuraphis noxia (Hemiptera: Aphididae). Salivary proteins were isolated and compared from three aphid probed diets: pure water, 15% sucrose, or amino acids (100 mM serine, 100 mM methionine, 100 mM aspartic acid, and 15% sucrose). After 6 h, more aphids settled on sucrose diet compared with other diets, but there were no signiÞcant differences in the number of stylet sheaths produced per aphid after 24 h. There were differences in the amount of soluble salivary protein (watery saliva), with the greatest amount secreted in sucrose diet, followed by amino acid diet and pure water, respectively. Protein constituents secreted into sucrose and amino acid diets were compared using gel electrophoresis using standardized amounts of protein. More protein bands and bands of greater intensity were visualized from probed sucrose diet compared with probed amino acid diet, indicating qualitative differences. Phosphatase was putatively identiÞed from D. noxia saliva from a major protein band using gel electrophoresis and mass spectrophotometry. Alkaline phosphatase activity was conÞrmed in sucrose diet using enzymatic assays but was not detected in aphid probed water or amino acid diets. Other peptides in sucrose diet weakly but signiÞcantly showed similarities to putative dehydrogenase and RNA helicase expressed sequence tags identiÞed from other aphids. The implications of these Þndings in aphid salivation and plantÐinsect interactions are discussed.
Comparisons of Salivary Proteins From Five Aphid (Hemiptera: Aphididae) Species
Environmental Entomology, 2011
Aphid (Hemiptera: Aphididae) saliva, when injected into host plants during feeding, causes physiological changes in hosts that facilitate aphid feeding and cause injury to plants. Comparing salivary constituents among aphid species could help identify which salivary products are universally important for general aphid feeding processes, which products are involved with speciÞc host associations, or which products elicit visible injury to hosts. We compared the salivary proteins from Þve aphid species, namely, Diuraphis noxia (Kurdjumov), D. tritici (Gillette), D. mexicana (Baker), Schizaphis graminum (Rondani), and Acyrthosiphon pisum (Harris). A 132-kDa protein band was detected from the saliva of all Þve species using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Alkaline phosphatase activity was detected from the saliva of all Þve species and may have a universal role in the feeding process of aphids. The Diuraphis species cause similar visible injury to grass hosts, and nine electrophoretic bands were unique to the saliva of these three species. S. graminum shares mutual hosts with the Diuraphis species, but visible injury to hosts caused by S. graminum feeding differs from that of Diuraphis feeding. Only two mutual electrophoretic bands were visualized in the saliva of Diuraphis and S. graminum. Ten unique products were detected from the saliva of A. pisum, which feeds on dicotyledonous hosts. Our comparisons of aphid salivary proteins revealed similarities among species which cause similar injury on mutual hosts, fewer similarities among species that cause different injury on mutual hosts, and little similarity among species which feed on unrelated hosts.
Identification of aphid salivary proteins: a proteomic investigation of Myzus persicae
Insect molecular biology, 2008
The role of insect saliva in the first contact between an insect and a plant is crucial during feeding. Some elicitors, particularly in insect regurgitants, have been identified as inducing plant defence reactions. Here, we focused on the salivary proteome of the green peach aphid, Myzus persicae. Proteins were either directly in-solution digested or were separated by 2D SDS-PAGE before trypsin digestion. Resulting peptides were then identified by mass spectrometry coupled with database investigations. A homemade database was constituted of expressed sequence tags from the pea aphid Acyrtosiphon pisum and M. persicae. The databases were used to identify proteins related to M. persicae with a nonsequenced genome. This procedure enabled us to discover glucose oxidase, glucose dehydrogenase, NADH dehydrogenase, α-glucosidase and α-amylase in M. persicae saliva. The presence of these enzymes is discussed in terms of plant–aphid interactions.
Journal of Proteome Research, 2011
The relationship between aphids and their host plants is thought to be functionally analogous to plant-pathogen interactions. Although virulence effector proteins that mediate plant defenses are well-characterized for pathogens such as bacteria, oomycetes, and nematodes, equivalent molecules in aphids and other phloem-feeders are poorly understood. A dual transcriptomic-proteomic approach was adopted to generate a catalog of candidate effector proteins from the salivary glands of the pea aphid, Acyrthosiphon pisum. Of the 1557 transcript supported and 925 mass spectrometry identified proteins, over 300 proteins were identified with secretion signals, including proteins that had previously been identified directly from the secreted saliva. Almost half of the identified proteins have no homologue outside aphids and are of unknown function. Many of the genes encoding the putative effector proteins appear to be evolving at a faster rate than homologues in other insects, and there is strong evidence that genes with multiple copies in the genome are under positive selection. Many of the candidate aphid effector proteins were previously characterized in typical phytopathogenic organisms (e.g., nematodes and fungi) and our results highlight remarkable similarities in the saliva from plant-feeding nematodes and aphids that may indicate the evolution of common solutions to the plant-parasitic lifestyle.
Journal of proteome research, 2015
Aphids deliver saliva into plants and acquire plant sap for their nourishment using a specialized mouthpart or stylets. Aphid saliva is of great importance because it contains effectors that are involved in modulating host defense and metabolism. Although profiling aphid salivary glands and identifying secreted proteins have been successfully used, success in direct profiling of aphid saliva have been limited due to scarcity of saliva collected in artificial diets. Here we present the use of a neurostimulant, resorcinol, for inducing aphid salivation. Saliva of potato aphids (Macrosiphum euphorbiae), maintained on tomato, was collected in resorcinol diet. Salivary proteins were identified using mass spectrometry and compared with the existing M. euphorbiae salivary proteome collected in water. Comparative analysis was also performed with existing salivary proteomes from additional aphid species. Most of the proteins identified in the resorcinol diet were also present in the water di...
ABSTRACT: Biotypes of aphids and many other insect pests are defined based on the phenotypic response of host plants to the insect pest without considering their intrinsic characteristics and genotypes. Plant breeders have spent considerable effort developing aphid-resistant, small-grain varieties to limit insecticide control of the greenbug, Schizaphis graminum. However, new S. graminum biotypes frequently emerge that break resistance. Mechanisms of virulence on the aphid side of the plant−insect interaction are not well understood. S. graminum biotype H is highly virulent on most small grain varieties. This characteristic makes biotype H ideal for comparative proteomics to investigate the basis of biotype virulence in aphids. In this study, we used comparative proteomics to identify protein expression differences associated with virulence. Aphid proteins involved in the tricarboxylic acid cycle, immune system, cell division, and antiapoptosis pathways were found to be up-regulated...
Proteomic profiling of cereal aphid saliva reveals both ubiquitous and adaptive secreted proteins
The secreted salivary proteins from two cereal aphid species, Sitobion avenae and Metopolophium dirhodum, were collected from artificial diets and analysed by tandem mass spectrometry. Protein identification was performed by searching MS data against the official protein set from the current pea aphid (Acyrthosiphon pisum) genome assembly and revealed 12 and 7 proteins in the saliva of S. avenae and M. dirhodum, respectively. When combined with a comparable dataset from A. pisum, only three individual proteins were common to all the aphid species; two paralogues of the GMC oxidoreductase family (glucose dehydrogenase; GLD) and ACYPI009881, an aphid specific protein previously identified as a putative component of the salivary sheath. Antibodies were designed from translated protein sequences obtained from partial cDNA sequences for ACYPI009881 and both saliva associated GLDs. The antibodies detected all parent proteins in secreted saliva from the three aphid species, but could only detect ACYPI009881, and not saliva associated GLDs, in protein extractions from the salivary glands. This result was confirmed by immunohistochemistry using whole and sectioned salivary glands, and in addition, localised ACYPI009881 to specific cell types within the principal salivary gland. The implications of these findings for the origin of salivary components and the putative role of the proteins identified are discussed in the context of our limited understanding of the functional relationship between aphid saliva and the plants they feed on. The mass spectrometry data have been deposited to the ProteomeXchange and can be accessed under the identifier PXD000113.
Proceedings of The National Academy of Sciences, 2008
In feeding, aphids inject saliva into plant tissues, gaining access to phloem sap and eliciting (and sometimes overcoming) plant responses. We are examining the involvement, in this aphid-plant interaction, of individual aphid proteins and enzymes, as identified in a salivary gland cDNA library. Here, we focus on a salivary protein we have arbitrarily designated Protein C002. We have shown, by using RNAi-based transcript knockdown, that this protein is important in the survival of the pea aphid (Acyrthosiphon pisum) on fava bean, a host plant. Here, we further characterize the protein, its transcript, and its gene, and we study the feeding process of knockdown aphids. The encoded protein fails to match any protein outside of the family Aphididae. By using in situ hybridization and immunohistochemistry, the transcript and the protein were localized to a subset of secretory cells in principal salivary glands. Protein C002, whose sequence contains an Nterminal secretion signal, is injected into the host plant during aphid feeding. By using the electrical penetration graph method on c002-knockdown aphids, we find that the knockdown affects several aspects of foraging and feeding, with the result that the c002-knockdown aphids spend very little time in contact with phloem sap in sieve elements. Thus, we infer that Protein C002 is crucial in the feeding of the pea aphid on fava bean. aphid-plant interaction ͉ saliva ͉ RNAi ͉ electrical penetration graph ͉ immunohistochemistry ‡ Present address:
Journal of Proteome Research, 2014
Biotypes of aphids and many other insect pests are defined based on the phenotypic response of host plants to the insect pest without considering their intrinsic characteristics and genotypes. Plant breeders have spent considerable effort developing aphid-resistant, small-grain varieties to limit insecticide control of the greenbug, Schizaphis graminum. However, new S. graminum biotypes frequently emerge that break resistance. Mechanisms of virulence on the aphid side of the plant-insect interaction are not well understood. S. graminum biotype H is highly virulent on most small grain varieties. This characteristic makes biotype H ideal for comparative proteomics to investigate the basis of biotype virulence in aphids. In this study, we used comparative proteomics to identify protein expression differences associated with virulence. Aphid proteins involved in the tricarboxylic acid cycle, immune system, cell division, and antiapoptosis pathways were found to be up-regulated in biotype H relative to other biotypes. Proteins from the bacterial endosymbiont of aphids were also differentially expressed in biotype H. Guided by the proteome results, we tested whether biotype H had a fitness advantage compared with other S. graminum biotypes and found that biotype H had a higher reproductive fitness as compared with two other biotypes on a range of different wheat germplasms. Finally, we tested whether aphid genetics can be used to further dissect the genetic mechanisms of biotype virulence in aphids. The genetic data showed that sexual reproduction is a source of biotypic variation observed in S. graminum.
Proteomic investigation of aphid honeydew reveals an unexpected diversity of proteins
PloS one, 2013
Aphids feed on the phloem sap of plants, and are the most common honeydew-producing insects. While aphid honeydew is primarily considered to comprise sugars and amino acids, its protein diversity has yet to be documented. Here, we report on the investigation of the honeydew proteome from the pea aphid Acyrthosiphon pisum. Using a two-Dimensional Differential in-Gel Electrophoresis (2D-Dige) approach, more than 140 spots were isolated, demonstrating that aphid honeydew also represents a diverse source of proteins. About 66% of the isolated spots were identified through mass spectrometry analysis, revealing that the protein diversity of aphid honeydew originates from several organisms (i.e. the host aphid and its microbiota, including endosymbiotic bacteria and gut flora). Interestingly, our experiments also allowed to identify some proteins like chaperonin, GroEL and Dnak chaperones, elongation factor Tu (EF-Tu), and flagellin that might act as mediators in the plant-aphid interactio...
Bacteria Associated with Russian Wheat Aphid (Diuraphis noxia) Enhance Aphid Virulence to Wheat
Phytobiomes journal, 2018
Phenotypic responses to biotic stresses are often studied as the interactions between two species; however, in the phytobiome, these responses frequently result from complex interactions involving several organisms. Here, we show that variation in chlorosis caused by Russian wheat aphid (Diuraphis noxia) feeding is determined, in part, by aphid-associated bacteria. Proteomic analysis of fluids injected into a sterile medium by the aphid during feeding indicate that 99% of the proteins are of bacterial origin. Of these, the greatest proportion are produced by bacteria in the order Enterobacteriales. Bacteria from five genera in four families that have the capacity to produce these proteins were isolated directly from aphids as well as from wheat leaves only after D. noxia feeding. By themselves or in combination, these bacteria were not virulent to wheat, even at high inoculum levels. Metagenomic analysis showed that the same five D. noxia-associated genera dominated the non-Buchnera component of the aphid microbiome, and that representation of these genera was reduced in aphids from colonies established after isolation of newborn nymphs from their mothers prior to feeding (isolated aphids). Isolation or treatment with antibiotics reduced bacterial numbers, and these aphids caused less feeding damage on wheat than non-isolated or non-antibiotic-treated aphids. Our data show that bacterial proteins are a significant component of Russian wheat aphid saliva, that the bacteria producing these proteins are associated with aphids and plants fed upon by aphids, and that these aphid-associated bacteria facilitate aphid virulence to wheat.
2014
The biochemical defence related responses conferred by Dn1 and Dn5 resistance genes in wheat against Russian wheat aphid (RWA) biotype 2 and biotype 3 in South Africa were investigated. The early responses of wheat to aphid infestation were determined and these included the activities of enzymes associated with the generation of reactive oxygen species (superoxide dismutase activity SOD and NADPH oxidase). The effect of aphid infestation on the activities of PR proteins was also determined. Biotype 2 and 3 infestation induced increase in enzyme activities of β-1,3-glucanase, peroxidase, NADPH oxidase and superoxide dismutase in the resistant wheat cultivar (PAN3144) hours post infestation, but a relatively lower enzyme activity of the uninfested plant of the resistant cultivar (PAN3144), susceptible uninfested cultivar (PAN3364) and the infested susceptible cultivar (PAN3364). These findings suggest that enzymes play a role as indicators in the RWA – wheat resistance response. The results obtained are consistent indicating that biotype 2 and biotypes 3 are virulent to Dn1 but, avirulent to Dn5 containing cultivars. Key words: Diuraphis noxia, biotypes, resistance, tolerance, wheat cultivars
Development of sample collection methods and preliminary identifications of aphid salivary proteins
The study of aphid salivary secretome has practical importance on understanding interactions of aphids and their host plants. Around 250 species of aphids out of the identified 4000 aphid species are considered as serious pests. The experiments were performed with pea aphids (Acyrthosiphon pisum) that were feeding on bean plants (Vivia fabe). Pea aphids feed on plant phloem sap by probing their stylet into the sieve elements of the plant and secreting saliva for external digestion. In order to collect aphid salivary proteins from the secreted saliva, small scale and large scale sample collection methods were carried out. The small scale sample method was performed in microfluidic devices using 10-25 aphids. Aphids were able to feed on the artificial diet by probing through a stretched Parafilm TM and survived for 2-3 days in the microfluidic devices. The experiments proved that the aphid survival and feeding rate could be improved with the factors such as ventilation, light intensity and increasing diet volume. However it was difficult to collect sufficient amounts of aphid saliva for detection using small scale devices. The large scale sample collection method was performed by feeding 8000 aphids in large screened chamber for 24/48h. The collected salivary samples after undergone a concentration process was capable of collecting detectable aphid salivary secretions. The experimental conditions were adjusted to obtain optimized HPLC separations. Finally, LC/MS/MS followed by peptide sequence database searching were able to identify potential aphid salivary proteins.
2016
Herbivore selection of plant hosts and plant responses to insect colonization have been subjects of intense investigations. A growing body of evidence suggests that for successful colonization to occur, (effector/virulence) proteins in insect saliva must modulate plant defense responses to the benefit of the insect. A range of insect saliva proteins that modulate plant defense responses have been identified, but there is no direct evidence that these proteins are delivered into specific plant tissues and enter plant cells. Aphids and other sap-sucking insects of the order Hemiptera use their specialized mouthparts (stylets) to probe plant mesophyll cells, until they reach the phloem cells for long-term feeding. Here we show by immunogold-labeling of ultrathin sections of aphid feeding sites that an immuno-suppressive aphid effector localizes in the cytoplasm of mesophyll cells near aphid stylets, but not in cells further away from aphid feeding sites. In contrast, another aphid effe...
PLoS Genetics, 2010
Aphids are amongst the most devastating sap-feeding insects of plants. Like most plant parasites, aphids require intimate associations with their host plants to gain access to nutrients. Aphid feeding induces responses such as clogging of phloem sieve elements and callose formation, which are suppressed by unknown molecules, probably proteins, in aphid saliva. Therefore, it is likely that aphids, like plant pathogens, deliver proteins (effectors) inside their hosts to modulate host cell processes, suppress plant defenses, and promote infestation. We exploited publicly available aphid salivary gland expressed sequence tags (ESTs) to apply a functional genomics approach for identification of candidate effectors from Myzus persicae (green peach aphid), based on common features of plant pathogen effectors. A total of 48 effector candidates were identified, cloned, and subjected to transient overexpression in Nicotiana benthamiana to assay for elicitation of a phenotype, suppression of the Pathogen-Associated Molecular Pattern (PAMP)-mediated oxidative burst, and effects on aphid reproductive performance. We identified one candidate effector, Mp10, which specifically induced chlorosis and local cell death in N. benthamiana and conferred avirulence to recombinant Potato virus X (PVX) expressing Mp10, PVX-Mp10, in N. tabacum, indicating that this protein may trigger plant defenses. The ubiquitin-ligase associated protein SGT1 was required for the Mp10-mediated chlorosis response in N. benthamiana. Mp10 also suppressed the oxidative burst induced by flg22, but not by chitin. Aphid fecundity assays revealed that in planta overexpression of Mp10 and Mp42 reduced aphid fecundity, whereas another effector candidate, MpC002, enhanced aphid fecundity. Thus, these results suggest that, although Mp10 suppresses flg22-triggered immunity, it triggers a defense response, resulting in an overall decrease in aphid performance in the fecundity assays. Overall, we identified aphid salivary proteins that share features with plant pathogen effectors and therefore may function as aphid effectors by perturbing host cellular processes.
PROTEOMICS, 2009
Nine proteins secreted in the saliva of the pea aphid Acyrthosiphon pisum were identified by a proteomics approach using GE-LC-MS/MS and LC-MS/MS, with reference to EST and genomic sequence data for A. pisum. Four proteins were identified by their sequences: a homolog of angiotensin-converting enzyme (an M2 metalloprotease), an M1 zinc-dependant metalloprotease, a glucose-methanol-choline (GMC)-oxidoreductase and a homolog to regucalcin (also known as senescence marker protein 30). The other five proteins are not homologous to any previously described sequence and included an abundant salivary protein (represented by ACYPI009881), with a predicted length of 1161 amino acids and high serine, tyrosine and cysteine content. A. pisum feeds on plant phloem sap and the metalloproteases and regucalcin (a putative calcium-binding protein) are predicted determinants of sustained feeding, by inactivation of plant protein defences and inhibition of calcium-mediated occlusion of phloem sieve elements, respectively. The amino acid composition of ACYPI009881 suggests a role in the aphid salivary sheath that protects the aphid mouthparts from plant defences, and the oxidoreductase may promote gelling of the sheath protein or mediate oxidative detoxification of plant allelochemicals. Further salivary proteins are expected to be identified as more sensitive MS technologies are developed.
Annals of The Entomological Society of America, 2000
Four hydrolases and Þve oxido-reductases were examined using native stacking polyacrylamide gel electrophoresis. Homogenate of Russian wheat aphid, Diuraphis noxia (Mordvilko), bird cherry-oat aphid, Rhopalosiphum padi (L.), ÔArapahoeÕ (aphid-susceptible) and ÔHaltÕ (aphid-resistant) wheat, Triticum aestivum L., and powdery mildewÐinfected Erysiphe graminis DC. ex Merat f. sp. tritici Em. Marchal, Arapahoe wheat leaves were assayed for enzyme activities. Pectinesterase, polygalacturonase (or pectinase), cellulase, and amylase activities were examined in the hydrolase group. Catalase, peroxidase, catechol oxidase, superoxide dismutase, and ascorbate oxidase activities were examined in the group of oxido-reductases. The two aphid species had the same hydrolases but different oxido-reductases. Although pectinesterase and cellulase enzymes were present in D. noxia and R. padi, the banding patterns were different. Polygalacturonase and ␦-amylase were not detected from either aphid species. In the oxido-reductase group, catalase was detected from D. noxia, wheras peroxidase was detected from R. padi. Superoxide dismutase and ascorbate oxidase activities also were detected from both aphids. Enzyme assays using aphid head tissue that included salivary glands but excluded aphid foregut supported the enzyme assays using whole aphids. Peroxidase activity was detected from the salivary tissue of R. padi, but not D. noxia, and catalase activity was detected from D. noxia salivary tissue, but not R. padi. We suggest that the salivary enzyme difference between the 2 aphid species (i.e., catalase and peroxidase) is important in the type of damage symptom formation on susceptible wheat plants.
Insect Biochemistry and Molecular Biology, 2008
Host insects are either susceptible or resistant to parasitoids, where resistant hosts express immunity factors and compatible parasitoids express virulence factors that may reveal the manipulation of susceptible hosts. Using proteomics we compared responses of the same host, the aphid Macrosiphum euphorbiae, challenged by a well-adapted parasitoid Aphidius nigripes or by a less adapted relative, Aphidius ervi. The host was found to be equally acceptable to both parasitoids, but while A. nigripes normally developed and killed hosts (high susceptibility), development of the incompatible A. ervi was arrested at the primary egg stage (high resistance). Two-dimensional gels at two stages of parasitism revealed divergence in patterns of protein regulation of the M. euphorbiae host, responding to A. ervi or A. nigripes, with the greatest number of protein modulations in the host resistance response. In A. erviresistant hosts, proPO was strongly up-regulated, as were also three cuticle proteins, suggesting a PO basis and exoskeleton reinforcement as early and late responses of M. euphorbiae to the risk of parasitism. Resistance also correlated with up-regulation of antioxidative, energy-related, cytoskeleton and heat shock proteins. In A. nigripes-susceptible hosts, various proteins implicated in host and bacterial symbiont metabolism were significantly altered, suggesting complex host nutritional modulation. Over-expression of energy-related proteins also increased when A. nigripes established and developed. Aphid proteomes of compatible and incompatible Aphidius parasitism provide an integrative basis for consolidating our knowledge of host-parasitoid interactions.
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Analysis of the Salivary Gland Transcriptome of Frankliniella occidentalis
PLoS ONE, 2014
Saliva is known to play a crucial role in insect feeding behavior and virus transmission. Currently, little is known about the salivary glands and saliva of thrips, despite the fact that Frankliniella occidentalis (Pergande) (the western flower thrips) is a serious pest due to its destructive feeding, wide host range, and transmission of tospoviruses. As a first step towards characterizing thrips salivary gland functions, we sequenced the transcriptome of the primary salivary glands of F. occidentalis using short read sequencing (Illumina) technology. A de novo-assembled transcriptome revealed 31,392 high quality contigs with an average size of 605 bp. A total of 12,166 contigs had significant BLASTx or tBLASTx hits (E#1.0E 26 ) to known proteins, whereas a high percentage (61.24%) of contigs had no apparent protein or nucleotide hits. Comparison of the F. occidentalis salivary gland transcriptome (sialotranscriptome) against a published F. occidentalis full body transcriptome assembled from Roche-454 reads revealed several contigs with putative annotations associated with salivary gland functions. KEGG pathway analysis of the sialotranscriptome revealed that the majority (18 out of the top 20 predicted KEGG pathways) of the salivary gland contig sequences match proteins involved in metabolism. We identified several genes likely to be involved in detoxification and inhibition of plant defense responses including aldehyde dehydrogenase, metalloprotease, glucose oxidase, glucose dehydrogenase, and regucalcin. We also identified several genes that may play a role in the extra-oral digestion of plant structural tissues including b-glucosidase and pectin lyase; and the extra-oral digestion of sugars, including a-amylase, maltase, sucrase, and a-glucosidase. This is the first analysis of a sialotranscriptome for any Thysanopteran species and it provides a foundational tool to further our understanding of how thrips interact with their plant hosts and the viruses they transmit. Citation: Stafford-Banks CA, Rotenberg D, Johnson BR, Whitfield AE, Ullman DE (2014) Analysis of the Salivary Gland Transcriptome of Frankliniella occidentalis. PLoS ONE 9(4): e94447.
Salivary proteins of plant-feeding hemipteroids – implication in phytophagy
Bulletin of Entomological Research, 2014
Many hemipteroids are major pests and vectors of microbial pathogens, infecting crops. Saliva of the hemipteroids is critical in enabling them to be voracious feeders on plants, including the economically important ones. A plethora of hemipteroid salivary enzymes is known to inflict stress in plants, either by degrading the plant tissue or by affecting their normal metabolism. Hemipteroids utilize one of the following three strategies of feeding behaviour: salivary sheath feeding, osmoticpump feeding and cell-rupture feeding. The last strategy also includes several different tactics such as lacerate-and-flush, lacerate-and-sip and macerate-and-flush. Understanding hemipteroid feeding mechanisms is critical, since feeding behaviour directs salivary composition. Saliva of the Heteroptera that are specialized as fruit and seed feeders, includes cell-degrading enzymes, auchenorrhynchan salivary composition also predominantly consists of cell-degrading enzymes such as amylase and protease, whereas that of the Sternorhyncha includes a variety of allelochemicaldetoxifying enzymes. Little is known about the salivary composition of the Thysanoptera. Cell-degrading proteins such as amylase, pectinase, cellulase and pectinesterase enable stylet entry into the plant tissue. In contrast, enzymes such as glutathione peroxidase, laccase and trehalase detoxify plant chemicals, enabling the circumvention of plant-defence mechanisms. Salivary enzymes such as M1-zinc metalloprotease and CLIP-domain serine protease as in Acyrthosiphon pisum (Aphididae), and non-enzymatic proteins such as apolipophorin, ficolin-3-like protein and 'lava-lamp' protein as in Diuraphis noxia (Aphididae) have the capacity to alter host-plant-defence mechanisms. A majority of the hemipteroids feed on phloem, hence Ca + + -binding proteins such as C002 protein, calreticulin-like isoform 1 and calmodulin (critical for preventing sieve-plate occlusion) are increasingly being recognized in hemipteroid-plant interactions. Determination of a staggering variety of proteins shows the complexity of hemipteroid saliva: effector proteins localized in hemipteran saliva suggest a similarity to the physiology of pathogen-plant interactions.
The genome of Diuraphis noxia, a global aphid pest of small grains
BMC genomics, 2015
The Russian wheat aphid, Diuraphis noxia Kurdjumov, is one of the most important pests of small grains throughout the temperate regions of the world. This phytotoxic aphid causes severe systemic damage symptoms in wheat, barley, and other small grains as a direct result of the salivary proteins it injects into the plant while feeding. We sequenced and de novo assembled the genome of D. noxia Biotype 2, the strain most virulent to resistance genes in wheat. The assembled genomic scaffolds span 393 MB, equivalent to 93% of its 421 MB genome, and contains 19,097 genes. D. noxia has the most AT-rich insect genome sequenced to date (70.9%), with a bimodal CpG(O/E) distribution and a complete set of methylation related genes. The D. noxia genome displays a widespread, extensive reduction in the number of genes per ortholog group, including defensive, detoxification, chemosensory, and sugar transporter groups in comparison to the Acyrthosiphon pisum genome, including a 65% reduction in che...
Intraspecific differences in plant defense induction by fall armyworm strains
The New phytologist, 2018
The underlying adaptive mechanisms by which insect strains are associated with specific plants are largely unknown. In this study, we investigated the role of herbivore-induced defenses in the host plant association of fall armyworm (Spodoptera frugiperda) strains. We tested the expression of herbivore-induced defense-related genes and the activity of plant-defensive proteins in maize and Bermuda grass upon feeding by fall armyworm strains. The rice strain caterpillars induced greater accumulation of proteinase inhibitors in maize than the corn strain caterpillars. In Bermuda grass, feeding by the corn strain suppressed induction of trypsin inhibitor activity whereas the rice strain induced greater activity levels. Differences in elicitation of these plant defenses by the two strains seems to be due to differences in the activity levels of the salivary enzyme phospholipase C. The levels of plant defense responses were negatively correlated with caterpillar growth, indicating a fitne...
PloS one, 2016
The Russian wheat aphid, Diuraphis noxia, an invasive phytotoxic pest of wheat, Triticum aestivum, and barley, Hordeum vulgare, causes huge economic losses in Africa, South America, and North America. Most acceptable and ecologically beneficial aphid management strategies include selection and breeding of D. noxia-resistant varieties, and numerous D. noxia resistance genes have been identified in T. aestivum and H. vulgare. North American D. noxia biotype 1 is avirulent to T. aestivum varieties possessing Dn4 or Dn7 genes, while biotype 2 is virulent to Dn4 and avirulent to Dn7. The current investigation utilized next-generation RNAseq technology to reveal that biotype 2 over expresses proteins involved in calcium signaling, which activates phosphoinositide (PI) metabolism. Calcium signaling proteins comprised 36% of all transcripts identified in the two D. noxia biotypes. Depending on plant resistance gene-aphid biotype interaction, additional transcript groups included those invol...
Gene expression plasticity across hosts of an invasive scale insect species
PloS one, 2017
For plant-eating insects, we still have only a nascent understanding of the genetic basis of host-use promiscuity. Here, to improve that situation, we investigated host-induced gene expression plasticity in the invasive lobate lac scale insect, Paratachardina pseudolobata (Hemiptera: Keriidae). We were particularly interested in the differential expression of detoxification and effector genes, which are thought to be critical for overcoming a plant's chemical defenses. We collected RNA samples from P. pseudolobata on three different host plant species, assembled transcriptomes de novo, and identified transcripts with significant host-induced gene expression changes. Gene expression plasticity was pervasive, but the expression of most detoxification and effector genes was insensitive to the host environment. Nevertheless, some types of detoxification genes were more differentially expressed than expected by chance. Moreover, we found evidence of a trade-off between expression of ...
Proteomic Insights into the Hidden World of Phloem Sap Feeding
Management of Insect Pests to Agriculture, 2016
The physical interface between a phloem-feeding insect and its host plant is a single cell buried deep within the plant tissue. As such, the molecular interactions between these notorious agricultural pests and the crop plants upon which they feed are diffi cult to study. 'Omic' technologies have proved crucial in revealing some of the fascinating detail of the molecular interplay between these partners. Here we review the role of proteomics in identifying putative components of the secreted saliva of phloem-feeding insects, particularly aphids, and discuss the limited knowledge concerning the function of these proteins.
Molecular & cellular proteomics : MCP, 2016
The two-spotted spider mite Tetranychus urticae is an extremely polyphagous crop pest. Alongside an unparalleled detoxification potential for plant secondary metabolites, it has recently been shown that spider mites can attenuate or even suppress plant defenses. Salivary constituents, notably effectors, have been proposed to play an important role in manipulating plant defenses and might determine the outcome of plant-mite interactions. Here, the proteomic composition of saliva from T. urticae lines adapted to various host plants - bean, maize, soy, and tomato - was analyzed using a custom-developed feeding assay coupled with nano-LC tandem mass spectrometry. About 90 putative T. urticae salivary proteins were identified. Many are of unknown function, and in numerous cases belonging to multi-membered gene families. RNAseq expression analysis revealed that many genes coding for these salivary proteins were highly expressed in the proterosoma, the mite body region that includes the sa...
Antibiosis to Metopolophium dirhodum (Homoptera: Aphididae) in Spring Wheat and Emmer Cultivars
Journal of Economic Entomology
Yield losses caused by pests, including aphids, can be substantial in cereals. Breeding for resistance against aphids is therefore desirable for enhancing the economic and environmental sustainability of cereal production. The aim of our study was to reveal the degree of antibiosis against Metopolophium dirhodum (Walker) (Homoptera: Aphididae), in four cultivars of spring wheat, Triticum aestivum L. (‘Alicia’, ‘Odeta’, ‘Libertina’, ‘Astrid’), and two cultivars of emmer, Triticum turgidum ssp. dicoccum (Schrank ex Schübler) Thell. (‘Rudico’, ‘Tapiruz’) (both Poales: Poaceae) under controlled laboratory conditions. Using age-stage, two-sex life table, we quantified responses of M. dirhodum to each cultivar and to project population growth. The spring wheat and emmer cultivars varied in their suitability to M. dirhodum. The cultivar most susceptible to M. dirhodum was the emmer cultivar ‘Rudico’; the projected population size of M. dirhodum on this cultivar was one order of magnitude l...
Aphid effector Me10 interacts with tomato TFT7, a 14-3-3 isoform involved in aphid resistance
New Phytologist
We demonstrated previously that expression of Macrosiphum euphorbiae salivary protein Me10 enhanced aphid reproduction on its host tomato (Solanum lycopersicum). However, the mechanism of action of Me10 remained elusive. To confirm the secretion of Me10 by the aphid into plant tissues, we produced Me10 polyclonal antibodies. To identify the plant targets of Me10, we developed a tomato immune induced complementary DNA yeast two-hybrid library and screened it with Me10 as bait. Immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays were performed to validate one of the interactions in planta, and virus-induced gene silencing was used for functional characterization in tomato. We demonstrated that Me10 is secreted into the plant tissues and interacts with tomato 14-3-3 isoform 7 (TFT7) in yeast. Immunoprecipitation assays confirmed that Me10 and its homologue in Aphis gossypii, Ag10k, interact with TFT7 in planta. Further, BiFC revealed that Me10 interaction with TFT7 occurs in the plant cell cytoplasm. While silencing of TFT7 in tomato leaves did not affect tomato susceptibility to M. euphorbiae, it enhanced longevity and fecundity of A. gossypii, the non-host aphid. Our results suggest the model whereby TFT7 plays a role in aphid resistance in tomato and effectors of the Me10/Ag10k family interfere with TFT7 function during aphid infestation.
Insects, 2020
Using host plant resistance (HPR) in management of insect pests is often environmentally friendly and suitable for sustainable development of agricultural industries. However, this strategy can be limited by rapid evolution of insect populations that overcome HPR, for which the underlying molecular factors and mechanisms are not well understood. To address this issue, we analyzed transcriptomes of two distinct biotypes of the grain aphid, Sitobion avenae (Fabricius), on wheat and barley. This analysis revealed a large number of differentially expressed genes (DEGs) between biotypes 1 and 3 on wheat and barley. The majority of them were common DEGs occurring on both wheat and barley. GO and KEGG enrichment analyses for these common DEGs demonstrated significant expression divergence between both biotypes in genes associated with digestion and defense. Top defense-related common DEGs with the most significant expression changes included three peroxidases, two UGTs (UDP-glycosyltransfe...
Comparative analyses of salivary proteins from three aphid species
Insect Molecular Biology, 2013
Saliva is a critical biochemical interface between aphids and their host plants; however, the biochemical nature and physiological functions of aphid saliva proteins are not fully elucidated. In this study we used a multidisciplinary proteomics approach combining liquid chromatography-electrospray ionization tandem mass spectrometry and two-dimensional differential in-gel electrophoresis/matrix-assisted laser desorption/ionization time-of-flight/mass spectrometry to compare the salivary proteins from three aphid species including Acyrthosiphon pisum, Megoura viciae and Myzus persicae. Comparative analyses revealed variability among aphid salivary proteomes. Among the proteins that varied, 22% were related to DNA-binding, 19% were related to GTP-binding, and 19% had oxidoreductase activity. In addition, we identified a peroxiredoxin enzyme and an ATP-binding protein that may be involved in the modulation of plant defences. Knowledge of salivary components and how they vary among aphid species may reveal how aphids target plant processes and how the aphid and host plant interact.
PLOS ONE, 2019
The successful parasitisation of a plant by a phytophagous insect is dependent on the delivery of effector molecules into the host. Sedentary gall forming insects, such as grape phylloxera (Daktulosphaira vitifoliae FITCH, Phylloxeridae), secrete multiple effectors into host plant tissues that alter or modulate the cellular and molecular environment to the benefit of the insect. The identification and characterisation of effector proteins will provide insight into the host-phylloxera interaction specifically the gall-induction processes and potential mechanisms of plant resistance. Using proteomic mass spectrometry and in-silico secretory prediction, 420 putative effectors were determined from the salivary glands or the root-feeding D. vitifoliae larvae reared on Teleki 5C (V. berlandieri x V. riparia). Among them, 170 conserved effectors were shared between D. vitifoliae and fourteen phytophagous insect species. Quantitative RT-PCR analysis of five conserved effector candidates (protein disulfideisomerase, peroxidoredoxin, peroxidase and a carboxypeptidase) revealed that their gene expression decreased, when larvae were starved for 24 h, supporting their assignment as effector molecules. The D. vitifoliae effectors identified here represent a functionally diverse group, comprising both conserved and unique proteins that provide new insight into the D. vitifoliae-Vitis spp. interaction and the potential mechanisms by which D. vitifoliae establishes the feeding site, suppresses plant defences and modulates nutrient uptake.
Arthropod-plant Interactions, 2017
Aphid saliva can suppress the blocking of sieve elements, a reaction that plants employ to inhibit aphid feeding, but aphid saliva can also elicit plant defence responses. Such plant responses might affect interactions between different aphid species and intraspecifically, e.g. among different biotypes. The objectives of our study were to investigate if feeding behaviour and performance of two biotypes of the lettuce aphid Nasonovia ribisnigri are affected by (1) feeding by the other biotype and (2) feeding by the green peach aphid Myzus persicae or the potato aphid Macrosiphum euphorbiae. Additionally the effect of feeding in a group was studied. All experiments were performed on both a resistant and an isogenic susceptible lettuce cultivar. Feeding or probing by conspecific or heterospecific aphids had different effects on Nasonovia ribisnigri biotypes. Aphids were only slightly affected by feeding or probing of the same biotype on both susceptible and resistant lettuce. N. ribisnigri virulent biotype Nr:1 suppressed the resistance against Nr:0 in the resistant cultivar. In contrast, defence was induced by Nr:1 against Nr:0 in susceptible lettuce. Co-infestation by M. euphorbiae and M. persicae had minor effects on Nr:0. Defence against Nr:1 was induced on both susceptible lettuce and resistant lettuce by Nr:0 and M. euphorbiae. Additionally, M. persicae induced defence in resistant lettuce against Nr:1. Effectors in the saliva of Nr:1 aphids are likely responsible for the defence suppression in lettuce. Identification of these effectors could lead to a better understanding of the mechanism of virulence in N. ribisnigri.
Vitellogenin from planthopper oral secretion acts as a novel effector to impair plant defenses
New Phytologist, 2021
Summary Vitellogenin (Vg) is a well‐known nutritious protein involved in reproduction in nearly all oviparous animals, including insects. Recently, Vg has been detected in saliva proteomes of several piercing–sucking herbivorous arthropods, including the small brown planthopper (Laodelphax striatellus, SBPH). Its function, however, remains unexplored. We investigated the molecular mechanism underlying SBPH orally secreted Vg‐mediated manipulation of plant–insect interaction by RNA interference, phytohormone and H2O2 profiling, protein–protein interaction studies and herbivore bioassays. A C‐terminal polypeptide of Vg (VgC) in SBPH, when secreted into rice plants, acted as a novel effector to attenuate host rice defenses, which in turn improved insect feeding performance. Silencing Vg reduced insect feeding and survival on rice. Vg‐silenced SBPH nymphs consistently elicited higher H2O2 production, a well‐established defense mechanism in rice, whereas expression of VgC in planta signi...
Insects, 2020
Grape phylloxera is an American native insect pest that caused heavy damages to the vineyards worldwide since its spreading to wine regions since the 1850s. This insect, able to feed on leaves and roots, induces plant galls and manipulates the grapevine physiology leading to plant damage and may cause plant death. The most successful treatment was the use of mostly partially resistant rootstocks. The degree of resistance is affected by environment, grapevine management and the insect biotype. In this study, we analyse the interaction of insect biotypes feeding on particular host plants. Therefore we evaluated the gene expression of Phylloxera feeding on a susceptible host versus feeding on a rootstock in two different developmental stages. We discovered (mainly in advanced insect developmental stages) genes expressed in higher proportion in one insect compared to the other. These genes related to chemosensory; in plant physiology manipulation and root deformation and insect digestive traits may play a role in the plant-insect interaction determining plant resistance in response to the pest attack.
Annals of Applied Biology, 2016
Crop protection is an integral part of establishing food security, by protecting the yield potential of crops. Cereal aphids cause yield losses by direct damage and transmission of viruses. Some wild relatives of wheat show resistance to aphids but the mechanisms remain unresolved. In order to elucidate the location of the partial resistance to the bird cherry-oat aphid, Rhopalosiphum padi, in diploid wheat lines of Triticum monococcum, we conducted aphid performance studies using developmental bioassays and electrical penetration graphs, as well as metabolic profiling of partially resistant and susceptible lines. This demonstrated that the partial resistance is related to a delayed effect on the reproduction and development of R. padi. The observed partial resistance is phloem based and is shown by an increase in number of probes before the first phloem ingestion, a higher number and duration of salivation events without subsequent phloem feeding and a shorter time spent phloem feeding on plants with reduced susceptibility. Clear metabolic phenotypes separate partially resistant and susceptible lines, with the former having lower levels of the majority of primary metabolites, including total carbohydrates. A number of compounds were identified as being at different levels in the susceptible and partially resistant lines, with asparagine, octopamine and glycine betaine elevated in less susceptible lines without aphid infestation. In addition, two of those, asparagine and octopamine, as well as threonine, glutamine, succinate, trehalose, glycerol, guanosine and choline increased in response to infestation, accumulating in plant tissue localised close to aphid feeding after 24 h. There was no clear evidence of systemic plant response to aphid infestation.