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• W3134159164 abstract "•Novel aphid bicycle genes contribute to plant gall development•Variation in a bicycle gene alters plant gene expression and a gall phenotype•bicycle genes encode a large family of diverse, secreted, cysteine-rich proteins•Many bicycle genes have experienced repeated diversifying selection In an elaborate form of inter-species exploitation, many insects hijack plant development to induce novel plant organs called galls that provide the insect with a source of nutrition and a temporary home. Galls result from dramatic reprogramming of plant cell biology driven by insect molecules, but the roles of specific insect molecules in gall development have not yet been determined. Here, we study the aphid Hormaphis cornu, which makes distinctive “cone” galls on leaves of witch hazel Hamamelis virginiana. We found that derived genetic variants in the aphid gene determinant of gall color (dgc) are associated with strong downregulation of dgc transcription in aphid salivary glands, upregulation in galls of seven genes involved in anthocyanin synthesis, and deposition of two red anthocyanins in galls. We hypothesize that aphids inject DGC protein into galls and that this results in differential expression of a small number of plant genes. dgc is a member of a large, diverse family of novel predicted secreted proteins characterized by a pair of widely spaced cysteine-tyrosine-cysteine (CYC) residues, which we named BICYCLE proteins. bicycle genes are most strongly expressed in the salivary glands specifically of galling aphid generations, suggesting that they may regulate many aspects of gall development. bicycle genes have experienced unusually frequent diversifying selection, consistent with their potential role controlling gall development in a molecular arms race between aphids and their host plants. In an elaborate form of inter-species exploitation, many insects hijack plant development to induce novel plant organs called galls that provide the insect with a source of nutrition and a temporary home. Galls result from dramatic reprogramming of plant cell biology driven by insect molecules, but the roles of specific insect molecules in gall development have not yet been determined. Here, we study the aphid Hormaphis cornu, which makes distinctive “cone” galls on leaves of witch hazel Hamamelis virginiana. We found that derived genetic variants in the aphid gene determinant of gall color (dgc) are associated with strong downregulation of dgc transcription in aphid salivary glands, upregulation in galls of seven genes involved in anthocyanin synthesis, and deposition of two red anthocyanins in galls. We hypothesize that aphids inject DGC protein into galls and that this results in differential expression of a small number of plant genes. dgc is a member of a large, diverse family of novel predicted secreted proteins characterized by a pair of widely spaced cysteine-tyrosine-cysteine (CYC) residues, which we named BICYCLE proteins. bicycle genes are most strongly expressed in the salivary glands specifically of galling aphid generations, suggesting that they may regulate many aspects of gall development. bicycle genes have experienced unusually frequent diversifying selection, consistent with their potential role controlling gall development in a molecular arms race between aphids and their host plants. Organisms often exploit individuals of other species, for example, through predation or parasitism. Parasites sometimes utilize molecular weapons against hosts, which themselves respond with molecular defenses, and the genes that encode or synthesize these molecular weapons may evolve rapidly in a continuous “arms race.”1Obbard D.J. Jiggins F.M. Halligan D.L. Little T.J. Natural selection drives extremely rapid evolution in antiviral RNAi genes.Curr. Biol. 2006; 16: 580-585Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 2Papkou A. Guzella T. Yang W. Koepper S. Pees B. Schalkowski R. Barg M.C. Rosenstiel P.C. Teotónio H. Schulenburg H. The genomic basis of Red Queen dynamics during rapid reciprocal host-pathogen coevolution.Proc. Natl. Acad. Sci. USA. 2019; 116: 923-928Crossref PubMed Scopus (0) Google Scholar, 3Paterson S. Vogwill T. Buckling A. Benmayor R. Spiers A.J. Thomson N.R. Quail M. Smith F. Walker D. Libberton B. et al.Antagonistic coevolution accelerates molecular evolution.Nature. 2010; 464: 275-278Crossref PubMed Scopus (321) Google Scholar Some of the most elaborate molecular defenses—such as adaptive immune systems, restriction modification systems, and CRISPR—have resulted from such host-parasite conflicts. In many less-well-studied systems, parasites not only extract nutrients from their hosts, but they also alter host behavior, physiology, or development to the parasite’s advantage.4Heil M. Host manipulation by parasites: cases, patterns, and remaining doubts.Front. Ecol. Evol. 2016; 4: 80Crossref Scopus (46) Google Scholar Insect galls represent one of the most extreme forms of such inter-species manipulation. Insect-induced galls are intricately patterned homes that provide insects with protection from environmental vicissitude and from some predators and parasites.5Bailey R. Schönrogge K. Cook J.M. Melika G. Csóka G. Thuróczy C. Stone G.N. Host niches and defensive extended phenotypes structure parasitoid wasp communities.PLoS Biol. 2009; 7: e1000179Crossref PubMed Scopus (0) Google Scholar, 6Mani M.S. Ecology of Plant Galls. Springer-Science+Business Media, 1964Crossref Google Scholar, 7Stone G.N. Schönrogge K. The adaptive significance of insect gall morphology.Trends Ecol. Evol. 2003; 18: 512-522Abstract Full Text Full Text PDF Scopus (513) Google Scholar Galls are also resource sinks, drawing nutrients from distant plant organs and providing insects with abundant food.8Larson K.C. Whitham T.G. Manipulation of food resources by a gall-forming aphid: the physiology of sink-source interactions.Oecologia. 1991; 88: 15-21Crossref PubMed Scopus (207) Google Scholar Insect galls are atypical plant growths that do not result simply from unpatterned cellular over-proliferation, as observed for microbial galls, like the crown gall induced by Agrobacterium tumefaciens. Instead, each galling insect species appears to induce a distinctive gall, even when related insect species attack the same plant, implying that each species provides unique instructions to reprogram latent plant developmental networks.9Cook L.G. Gullan P.J. Insect, not plant, determines gall morphology in the Apiomorpha pharetrata species-group (Hemiptera: Coccoidea).Aust. J. Entomol. 2008; 47: 51-57Crossref Scopus (10) Google Scholar, 10Crespi B. Worobey M. Comparative analysis of gall morphology in Australian gall thrips: The evolution of extended phenotypes.Evolution. 1998; 52: 1686-1696Crossref PubMed Google Scholar, 11Dodson G.N. Control of gall morphology: tephritid gallformers (Aciurina spp.) on rabbitbrush (Chrysothamnus).Ecol. Entomol. 1991; 16: 177-181Crossref Scopus (8) Google Scholar, 12Hearn J. Blaxter M. Schönrogge K. Nieves-Aldrey J.-L. Pujade-Villar J. Huguet E. Drezen J.-M. Shorthouse J.D. Stone G.N. Genomic dissection of an extended phenotype: oak galling by a cynipid gall wasp.PLoS Genet. 2019; 15: e1008398Crossref PubMed Scopus (19) Google Scholar, 13Leatherdale D. Plant hyperplasia induced with a cell-free insect extract.Nature. 1955; 175: 553-554Crossref Scopus (9) Google Scholar, 14Martin J.P. Stem galls of sugar cane induced with an insect extract.Hawaii. Plant. Rec. 1938; 42: 129-134Google Scholar, 15Martinson E. Werren J. Egan S. Tissue-specific gene expression shows cynipid wasps repurpose host gene networks to create complex and novel parasite-specific organs on oaks.Authorea. 2020; https://doi.org/10.22541/au.159466932.21772181Crossref Google Scholar, 16Parr T. Asterolecanium variolosum Ratzeburg, a gall-forming coccid, and its effect upon the host trees.Yale University School of Forestry Bulletin. 1940; 46: 1-49Google Scholar, 17Plumb G.H. The formation and development of the Norway Spruce gall caused by Adelges abietis L.Bulletin of the Connecticut Experiment Station. 1953; 566: 1-77Google Scholar, 18Stern D.L. Phylogenetic evidence that aphids, rather than plants, determine gall morphology.Proc. R. Soc. Lond. B Biol. Sci. 1995; 260: 85-89Crossref Google Scholar, 19Stone G.N. Cook J.M. The structure of cynipid oak galls: patterns in the evolution of an extended phenotype.Proc. Biol. Sci. 1998; 265: 979-988Crossref Scopus (0) Google Scholar At least some gall-inducing insects produce phytohormones,20Dorchin N. Hoffmann J.H. Stirk W.A. Novák O. Strnad M. Van Staden J. Sexually dimorphic gall structures correspond to differential phytohormone contents in male and female wasp larvae.Physiol. Entomol. 2009; 34: 359-369Crossref Scopus (28) Google Scholar, 21McCalla D.R. Genthe M.K. Hovanitz W. Chemical nature of an insect gall growth-factor.Plant Physiol. 1962; 37: 98-103Crossref PubMed Google Scholar, 22Suzuki H. Yokokura J. Ito T. Arai R. Yokoyama C. Toshima H. Nagata S. Asami T. Suzuki Y. Biosynthetic pathway of the phytohormone auxin in insects and screening of its inhibitors.Insect Biochem. Mol. Biol. 2014; 53: 66-72Crossref PubMed Scopus (19) Google Scholar, 23Tanaka Y. Okada K. Asami T. Suzuki Y. Phytohormones in Japanese mugwort gall induction by a gall-inducing gall midge.Biosci. Biotechnol. Biochem. 2013; 77: 1942-1948Crossref PubMed Scopus (23) Google Scholar, 24Tooker J.F. Helms A.M. Phytohormone dynamics associated with gall insects, and their potential role in the evolution of the gall-inducing habit.J. Chem. Ecol. 2014; 40: 742-753Crossref PubMed Scopus (62) Google Scholar, 25Yamaguchi H. Tanaka H. Hasegawa M. Tokuda M. Asami T. Suzuki Y. Phytohormones and willow gall induction by a gall-inducing sawfly.New Phytol. 2012; 196: 586-595Crossref PubMed Scopus (58) Google Scholar although it is not yet clear whether insects introduce these hormones into plants to support gall development. However, injection of phytohormones alone probably cannot generate the large diversity of species-specific insect galls. In addition, galling insects induce plant transcriptional changes independently of phytohormone activity.2Papkou A. Guzella T. Yang W. Koepper S. Pees B. Schalkowski R. Barg M.C. Rosenstiel P.C. Teotónio H. Schulenburg H. The genomic basis of Red Queen dynamics during rapid reciprocal host-pathogen coevolution.Proc. Natl. Acad. Sci. USA. 2019; 116: 923-928Crossref PubMed Scopus (0) Google Scholar,12Hearn J. Blaxter M. Schönrogge K. Nieves-Aldrey J.-L. Pujade-Villar J. Huguet E. Drezen J.-M. Shorthouse J.D. Stone G.N. Genomic dissection of an extended phenotype: oak galling by a cynipid gall wasp.PLoS Genet. 2019; 15: e1008398Crossref PubMed Scopus (19) Google Scholar,26Bailey S. Percy D.M. Hefer C.A. Cronk Q.C.B. The transcriptional landscape of insect galls: psyllid (Hemiptera) gall formation in Hawaiian Metrosideros polymorpha (Myrtaceae).BMC Genomics. 2015; 16: 943Crossref PubMed Scopus (17) Google Scholar, 27Nabity P.D. Haus M.J. Berenbaum M.R. DeLucia E.H. Leaf-galling phylloxera on grapes reprograms host metabolism and morphology.Proc. Natl. Acad. Sci. USA. 2013; 110: 16663-16668Crossref PubMed Scopus (75) Google Scholar, 28Shih T.H. Lin S.H. Huang M.Y. Sun C.W. Yang C.M. Transcriptome profile of cup-shaped galls in Litsea acuminata leaves.PLoS ONE. 2018; 13: e0205265Crossref PubMed Scopus (9) Google Scholar, 29Takeda S. Yoza M. Amano T. Ohshima I. Hirano T. Sato M.H. Sakamoto T. Kimura S. Comparative transcriptome analysis of galls from four different host plants suggests the molecular mechanism of gall development.PLoS ONE. 2019; 14: e0223686Crossref PubMed Scopus (4) Google Scholar Thus, given the complex cellular changes required for gall development, insects probably introduce many molecules into plant tissue to induce galls. In addition to the potential role of phytohormones in promoting gall growth, candidate gall effectors have been identified in several gall-forming insects.30Cambier S. Ginis O. Moreau S.J.M. Gayral P. Hearn J. Stone G.N. Giron D. Huguet E. Drezen J.-M. Gall wasp transcriptomes unravel potential effectors involved in molecular dialogues with oak and rose.Front. Physiol. 2019; 10: 926Crossref PubMed Scopus (9) Google Scholar, 31Eitle M.W. Carolan J.C. Griesser M. Forneck A. The salivary gland proteome of root-galling grape phylloxera (Daktulosphaira vitifoliae Fitch) feeding on Vitis spp.PLoS ONE. 2019; 14: e0225881Crossref PubMed Scopus (8) Google Scholar, 32Zhao C. Escalante L.N. Chen H. Benatti T.R. Qu J. Chellapilla S. Waterhouse R.M. Wheeler D. Andersson M.N. Bao R. et al.A massive expansion of effector genes underlies gall-formation in the wheat pest Mayetiola destructor.Curr. Biol. 2015; 25: 613-620Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar However, none of these candidate effectors have yet been shown to contribute to gall development or physiology. In addition, although many herbivorous insects introduce effector molecules into plants to influence plant physiology,33Elzinga D.A. Jander G. The role of protein effectors in plant-aphid interactions.Curr. Opin. Plant Biol. 2013; 16: 451-456Crossref PubMed Scopus (84) Google Scholar, 34Hogenhout S.A. Bos J.I.B. Effector proteins that modulate plant--insect interactions.Curr. Opin. Plant Biol. 2011; 14: 422-428Crossref PubMed Scopus (0) Google Scholar, 35Kaloshian I. Walling L.L. Hemipteran and dipteran pests: effectors and plant host immune regulators.J. Integr. Plant Biol. 2016; 58: 350-361Crossref PubMed Scopus (45) Google Scholar, 36Stuart J. Insect effectors and gene-for-gene interactions with host plants.Curr. Opin. Insect Sci. 2015; 9: 56-61Crossref PubMed Scopus (38) Google Scholar there is no evidence that any previously described effectors contribute to gall development. Because there are currently no galling insect model systems that would facilitate a genetic approach to this problem, we turned to natural variation to identify insect genes that contribute to gall development. We studied the aphid, Hormaphis cornu, which induces galls on the leaves of witch hazel, Hamamelis virginiana, in the Eastern United States (Figures 1A–1F and 1J ). In early spring, each H. cornu gall foundress (fundatrix) probes an expanding leaf with her microscopic mouthparts (stylets; Figures 1A and 1B; Video S1) and pierces individual mesophyll cells with her stylets (Figures 1G and 1H).37Lewis I.F. Walton L. Initiation of the cone gall of witch hazel.Science. 1947; 106: 419-420Crossref PubMed Scopus (1) Google Scholar,38Lewis I.F. Walton L. Gall-formation on Hamamelis virginiana resulting from material injected by the aphid Hormaphis hamamelidis.Trans. Am. Microsc. Soc. 1958; 77: 146-200Crossref Google Scholar We found that plant cells near injection sites, revealed by the persistent stylet sheaths, over-proliferate through periclinal cell divisions (Figure 1H). This pattern of cytokinesis is not otherwise observed in leaves at this stage of development (Figure 1I) and contributes to the thickening and expansion of leaf tissue that generates the gall (Figures 1D–1G). The increased proliferation of cells near the tips of stylet sheaths suggests that secreted effector molecules produced in the salivary glands are deposited into the plant via the stylets. https://www.cell.com/cms/asset/8f8c5743-5906-46f0-b994-3c9cf9d04143/mmc2.mp4Loading ... Download .mp4 (47.07 MB) Help with .mp4 files Video S1. Description of the phenomenon of insect gall development, including time-lapse video of H. cornu fundatrices directing gall formation on budding leaves of H. virginiana, related to Figure 1 After several days, the basal side of the gall encloses the fundatrix and the gall continues to grow apically and laterally, providing the fundatrix and her offspring with protection and abundant food. After several weeks, the basal side of the gall opens to allow aphids to remove excreta (honeydew) and molted nymphal skins from the gall and, eventually, to allow winged migrants to depart. Continued gall growth requires the constant presence of the fundatrix and gall tissue dies in her absence,38Lewis I.F. Walton L. Gall-formation on Hamamelis virginiana resulting from material injected by the aphid Hormaphis hamamelidis.Trans. Am. Microsc. Soc. 1958; 77: 146-200Crossref Google Scholar,39Rehill B.J. Schultz J.C. Hormaphis hamamelidis and gall size: a test of the plant vigor hypothesis.Oikos. 2001; 95: 94-104Crossref Scopus (24) Google Scholar suggesting that the fundatrix must continuously inject salivary-gland-produced effectors to overcome plant defenses. We found that populations of H. cornu galls include approximately 4% red galls and 96% green galls (Figure 1F). We inferred that this gall color polymorphism results from differences among aphids, rather than from differences associated with leaves or the location of galls on leaves, because red and green galls are located randomly on leaves and often adjacent to each other on a single leaf (Figure 1F). We sequenced and annotated the genome of H. cornu (Figures S1A and S1B; STAR methods) and performed a genome-wide association study (GWAS) on fundatrices isolated from 43 green galls and 47 red galls by resequencing their genomes to approximately 3× coverage. There is no evidence for genome-wide differentiation of samples from red and green galls, suggesting that individuals making red and green galls were sampled from a single interbreeding population (Figures S1C–S1F). We identified SNPs near 40.5 Mbp on chromosome 1 that were strongly associated with gall color (Figure 2A). We resequenced approximately 800 kbp flanking these SNPs to approximately 60× coverage and identified 11 SNPs within the introns and upstream of gene g16073 that were strongly associated with gall color (Figures 2B–2D). There is no evidence that large-scale chromosomal aberrations are associated with gall color (Figures S1G–S1K; STAR methods). Because GWASs can sometimes produce spurious associations, we performed an independent replication study and found that all 11 SNPs were highly significantly associated with gall color in fundatrices isolated from 435 green and 431 red galls (LOD = 191–236; Figure 2E). All fundatrices from green galls were homozygous for the ancestral allele at 9 or more of these SNPs (Figure 2E). In contrast, 98% of fundatrices from red galls were heterozygous or homozygous for derived alleles at 9 or more SNPs (Figure 2E). This pattern suggests that alleles contributing to red gall color are genetically dominant to alleles that generate green galls. Two percent of fundatrices that induce red galls were homozygous for ancestral alleles at these SNPs and likely carry genetic variants elsewhere in the genome that confer red color to galls (Figure S1L; STAR methods). Based on these genetic associations and further evidence presented below, we assigned the name determinant of gall color (dgc) to g16073. dgc encodes a predicted protein of 23 kDa with an N-terminal secretion signal sequence (Figure S1M). The putatively secreted portion of the protein shares no detectable sequence homology with any previously reported proteins. Most SNPs associated with green or red galls were found in one of two predominant haplotypes (Figure 2E) and exhibited strong linkage disequilibrium (LD) (Figures S2A–S2D). LD can result from suppressed recombination. However, these 11 SNPs are in linkage equilibrium with many other intervening and flanking SNPs (Figure S2). Also, multiple observed genotypes are consistent with recombination between these 11 SNPs (Figure 2E), and we found no evidence for chromosomal aberrations that could suppress recombination (Figures S1G–S1K; STAR methods). Thus, LD among the 11 dgc SNPs associated with gall color cannot be explained by suppressed recombination. It is more likely that the non-random association of the 11 dgcRed SNPs has been maintained by natural selection, suggesting that the combined action of all 11 SNPs may have a stronger effect on gene function than any single SNP alone. Because all 11 dgc polymorphisms associated with gall color occur outside of dgc exons (Figure 2D), we tested whether these polymorphisms influence expression of dgc or of any other genes in the genome. We first determined that dgc is expressed highly and specifically in fundatrix salivary glands and lowly or not at all in other tissues or other life cycle stages (Figure 3A). We then performed RNA sequencing (RNA-seq) on salivary glands from fundatrices with dgcGreen/dgcGreen or dgcRed/dgcGreen genotypes. dgc stands out as the most strongly differentially expressed gene between these genotypes (Figure 3B). Because dgcRed alleles appeared to be dominant to the dgcGreen alleles for gall color, we expected that dgc transcripts would be upregulated in animals with dgcRed alleles. In contrast, dgc transcripts were almost absent in fundatrices carrying dgcRed alleles (Figure 3C). That is, red galls are associated with strongly reduced dgc expression in fundatrix salivary glands. dgc expression is reduced approximately 20-fold in fundatrix salivary glands with dgcRed/dgcGreen (27 ± 22.6 cpm; mean ± SD) versus dgcGreen/dgcGreen genotypes (536 ± 352.3 cpm; mean ± SD). This result suggested that dgcRed alleles downregulate both the dgcRed and dgcGreen alleles in heterozygotes. To confirm whether the dgcRed allele downregulates the dgcGreen allele in trans, we identified exonic SNPs that were specific to each allele and could be identified in the RNA-seq data. We found that both dgcRed and dgcGreen alleles were strongly downregulated in heterozygotes, confirming the trans activity of the dgcRed allele (Figure S3A). We observed no systematic transcriptional changes in neighboring genes (Figure 3C), most of which are dgc paralogs, indicating that dgcRed alleles exhibit a perhaps unique example of locus-specific repressive transvection.40Duncan I.W. Transvection effects in Drosophila.Annu. Rev. Genet. 2002; 36: 521-556Crossref PubMed Scopus (167) Google Scholar Because red galls are associated with strong differential expression of only dgc, we wondered how the plant responds to changes in this single putative effector. To examine this question, we sequenced and annotated the genome of the host plant Hamamelis virginiana and then performed whole-genome differential expression on plant mRNA isolated from galls induced by aphids with dgcRed/dgcGreen versus dgcGreen/dgcGreen genotypes (STAR methods). We did not observe genome-wide differentiation between red and green galls (Figures S3B and S3C), only eight plant genes were differentially expressed between red and green galls, and all eight genes were downregulated in green galls (Figures 4A and 4B ). That is, high levels of dgc are associated with downregulation of only eight plant genes in galls. Red pigmented galls could result from production of carotenoids,41Smits B.L. Peterson W.J. Carotenoids of telial galls of Gymnosporangium Juniperi-Virginianae Lk.Science. 1942; 96: 210-211Crossref PubMed Scopus (0) Google Scholar anthocyanins,42Blunden G. Challen S.B. Red pigment in leaf galls of Salix fragilis L.Nature. 1965; 208: 388-389Crossref Scopus (2) Google Scholar,43Bomfim P.M.S. Cardoso J.C.F. Rezende U.C. Martini V.C. Oliveira D.C. Red galls: the different stories of two gall types on the same host.Plant Biol. 2019; 21: 284-291Crossref PubMed Scopus (4) Google Scholar or betacyanins. However, in red galls induced by H. cornu, the seven most strongly upregulated plant genes are all homologous to genes annotated as enzymes of the anthocyanin biosynthetic pathway (Figure 4C). One gene encodes an enzyme (ACCA) that irreversibly converts acetyl-coenzyme A (CoA) to malonyl CoA, a biosynthetic precursor of multiple anthocyanins. Two genes encode anthocyanidin 3-0-glucosyltransferases (UFGT and UGT75C1), which glycosylate unstable anthocyanidins to allow their accumulation.44Springob K. Nakajima J. Yamazaki M. Saito K. Recent advances in the biosynthesis and accumulation of anthocyanins.Nat. Prod. Rep. 2003; 20: 288-303Crossref PubMed Scopus (264) Google Scholar Two genes encode flavonoid 3′-5′ methyltransferases (FAOMT-1 and FAOMT-2), which methylate anthocyanin derivatives.45Hugueney P. Provenzano S. Verriès C. Ferrandino A. Meudec E. Batelli G. Merdinoglu D. Cheynier V. Schubert A. Ageorges A. A novel cation-dependent O-methyltransferase involved in anthocyanin methylation in grapevine.Plant Physiol. 2009; 150: 2057-2070Crossref PubMed Scopus (0) Google Scholar Finally, two genes encode phi class glutathione S-transferases (GSTF11 and GSTF12), which conjugate glutathione to anthocyanins, facilitating anthocyanin transport and stable accumulation in vacuoles.46Marrs K.A. Alfenito M.R. Lloyd A.M. Walbot V. A glutathione S-transferase involved in vacuolar transfer encoded by the maize gene Bronze-2.Nature. 1995; 375: 397-400Crossref PubMed Google Scholar Six of the enzymes upregulated in red galls are required for final steps of anthocyanin production and deposition (Figure 4C), and their upregulation in red galls may account for the accumulation of pigments in red galls. To test this hypothesis, we extracted and analyzed pigments from galls (Figure 4D) and identified high levels of two pigments only in red galls (Figure 4E), the anthocyanins malvidin-3,5-diglucoside and peonidin-3,5-diglucoside (Figures 4F and S3F–S3J). Thus, the pigments present in red galls are products of enzymes in the anthocyanin biosynthetic pathway, such as those encoded by genes that are upregulated in red galls. The two abundant anthocyanins are produced from distinct intermediate precursor molecules (Figure 4C), three of which were also detected in red galls (Figures 4F, S3F, S3G, and S3I), and synthesis of these two anthocyanins likely requires activity of different methyltransferases and glucosyltransferases. The three pairs of glucosyltransferases, methyltransferases, and glutathione transferases upregulated in red galls may provide the specific activities required for production of these two anthocyanins. Taken together, these observations suggest that dgc represses transcription of seven anthocyanin biosynthetic enzymes. It is not clear how dgc induces specific transcriptional changes in seven plant genes; it may act by altering activity of an upstream regulator of these plant genes. Gall color represents only one aspect of the gall phenotype, apparently mediated by changes in expression of seven plant genes, and the full complement of cell biological events during gall development presumably requires changes in many more plant genes. To estimate how many plant genes are differentially expressed during development of the H. cornu gall on H. virginiana, we performed differential expression analysis of plant genes in galls versus the surrounding leaf tissue. Approximately 31% of plant genes were upregulated and 34% were downregulated at false discovery rate (FDR) = 0.05 in galls versus leaf tissue (Figure 4G; 27% up and 29% down in gall at FDR = 0.01). Results of Gene Ontology analysis of up and downregulated genes are consistent with the extensive growth of gall tissue and downregulation of chloroplasts seen in aphid galls (Figure 4H; STAR methods), a pattern observed in other galling systems.12Hearn J. Blaxter M. Schönrogge K. Nieves-Aldrey J.-L. Pujade-Villar J. Huguet E. Drezen J.-M. Shorthouse J.D. Stone G.N. Genomic dissection of an extended phenotype: oak galling by a cynipid gall wasp.PLoS Genet. 2019; 15: e1008398Crossref PubMed Scopus (19) Google Scholar Thus, approximately 15,000 plant genes are differentially expressed in galls, representing a system-wide reprogramming of plant cell biology. If other aphid effector molecules act in ways similar to dgc, which is associated with differential expression of only eight plant genes, then gall development may require injection of hundreds or thousands of effector molecules. To identify additional proteins that aphids may inject into plants to contribute to gall development, we exploited the fact that only some individuals in the complex life cycle of H. cornu induce galls (Figure 1J). Only the fundatrix generation induces galls, and only her immediate offspring live alongside her in the developing gall. In contrast, individuals of generations that live on river birch (Betula nigra) through the summer and the sexual generation that feed on H. virginiana leaves in the autumn do not induce any leaf malformations. Thus, probably only the salivary glands of the generations that induce galls (the fundatrix [G1] and possibly also her immediate offspring [G2]) produce gall-effector molecules. We identified 3,048 genes upregulated in fundatrix salivary glands versus the fundatrix body (Figure S4A) and 3,427 genes upregulated in salivary glands of fundatrices, which induce galls, versus sexuals, which do not induce galls, although they feed on the same host plant (Figure S4B). Intersection of these gene sets identified 1,482 genes specifically enriched in the salivary glands of fundatrices (Figure S4C). Half of these genes (744) were homologous to previously identified genes, many of which had functional annotations (Figure 5A). Gene Ontology analysis of the “annotated” genes suggests that they contribute mostly to the demands for high levels of protein secretion in fundatrix salivary glands (Figure S4D). Most do not encode proteins with secretion signals (671; Figure S4E) and are thus unlikely to be injected into plants. We searched for homologs of genes that have been proposed as candidate gall-effector genes in other insects but found little evidence that these classes of genes contribute to aphid gall development (Figures S4F–S4H). We therefore focused on t" @default.
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• W3134159164 title "A novel family of secreted insect proteins linked to plant gall development" @default.
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