- Secretory RING finger proteins function as effectors in a grapevine galling insect. - fundamental to growth, development, or perception of the environment, leading to complex but often predictable responses to stress. - As a specialized component of the ubiquitin-proteasome system (UPS), the RING finger domain mediates protein-protein interactions and displays considerable versatility in regulating many physiological. - Moreover, the majority of the SPRINGs were more expressed in the feeding stage than the non-feeding egg stage, in contrast to the non-secretory RING genes.. - An insect SPRING interacted with two plant proteins, a cellulose synthase, CSLD5, and a ribosomal protein, RPS4B suggesting secretion reprograms host immune signaling, cell division, and stress response in favor of the insect. - Numerous reviews highlight what component processes of the UPS serve as targets for pathogens (e.g., [5, 6. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Full list of author information is available at the end of the article. - E3 genes typically make up the largest gene families in plants with elevated rates of evolution, reflecting their broad regulation of specific substrates, and also show en- richment in intronless genes, an artifact of the evolution of genes that function in rapid turnover [13–15]. - vitifoliae, is a pest of cultivated grapevine, with a nearly worldwide distribution, following its accidental introduction from Northern America to the rest of the world. - vitifoliae to the Old World wreaked havoc on the culture of the cul- tivated vine, Vitis vinifera. - vitifoliae encodes a large number of secretory RING finger protein genes. - In this study, we developed a bioinformatics pipeline that incorporated both transcriptome and genome sequences to predict non-secretory RING finger proteins and SPRINGs in D. - Among these, 138 were predicted as SPRINGs for the presence of signal peptides and absence of transmembrane domains, and the other 106 were predicted as non-secretory RING proteins for lacking signal peptides or containing transmembrane do- mains (Fig. - SPRINGs were small-sized and evolutionarily non- conserved, relative to the non-secretory proteins. - Comparisons of SPRINGs and non-secretory RING pro- teins revealed that SPRINGs (median size = 252 aa) were significantly smaller (one-tailed unpaired t-test p-value. - 0.01) than the non-secretory proteins (median size = 538 aa. - Sequence homology searches (BLASTP) indicated that the secretory RING proteins mostly showed little to no sequence similarity to the known proteins deposited in the GenBank databases, in contrast to the non-secretory proteins, most of which were highly similar to known proteins. - The median top hit e-value was 1e-4 for secretory proteins and 1e-180 for non- secretory proteins (Fig. - In addition, compared to the non-secretory RING proteins that were most similar to their Aphididae homologs (104 top BLAST hit species are aphids), the secretory RING proteins were mostly similar to non-insect species (71 top hits), or specific to D. - Overall, thus, this shows a stark con- trast between non-secretory RINGs, which almost always have homologs in aphid, with high conservation of se- quences, and secretory RINGs which are often no-hit or at least have very low levels of sequence conservation.. - Molecular rate analysis of gene families within secretory and non-secretory RINGs also showed that the nonsy- nonymous to synonymous substitution rate ratio (dN/. - 0.01) in the secretory proteins (median = 0.57) than in the non-secretory proteins (median = 0.05). - Secretory RING protein genes are more likely to duplicate and express at feeding stage. - viti- foliae, we constructed the Bayesian inference of phylogen- etic trees separately for secretory and no-secretory RING proteins because of the sequence divergence between these two groups. - Based on a 0.9 posterior probabilities threshold to cluster ≥3 RING proteins, only six (6%) non- secretory proteins formed two clusters, NSE-1 and NSE-2 (Fig. - Even the clustering thresholds were lowered to 0.5 posterior probabilities for ≥2 RING proteins, the ma- jority (75, or 71%) of the non-secretory RING proteins still existed as singletons. - However, in the secretory group, 98 (71%) RING proteins formed 10 clusters (SCE-1 through SCE-10) with the largest including 18 members, based on the stringent clustering threshold used above (Fig. - When using the loose threshold of secretory RING proteins formed clusters. - In addition, we observed that clusters in the secretory RING group were often formed by genes adjacent in the genome sequence, for ex- ample, 11 SCE-5 genes were located in scaffold #534, sug- gesting a pattern of recurrent tandem duplication that. - 1 Bioinformatics pipeline to identify putatively secretory and non-secretory RING finger proteins from D. - b Selection of secretory and non-secretory RING finger proteins. - caused the expansion of secretory RING genes in D. - Given that most of these secretory RING pro- teins showed little to no sequence similarity to other known proteins, they are likely to have multiplied in the insect genome through species-specific gene duplication.. - RNA-Seq-based expression analyses indicated that the secretory and non-secretory RING protein genes displayed strong expression profiles distinct to each insect life stage and fewer genes specific to location. - Out of the 124 tested secretory protein genes, 94 (75%) were higher expressed in adults than in eggs, and only 2 were higher expressed in eggs than in adults (Fig. - out of 103 tested non-secretory protein genes were higher expressed in adults than in eggs, and the majority (88, or 85%) were similarly expressed in both life stages. - Notably, expression of 9 (8%) non-secretory protein genes were even upregulated in eggs in relative to adults (Fig. - Of these genes 16 secretory and 3 non-secretory were expressed higher in North America origin individuals whereas 6 genes of each type were expressed higher in France origin individ- uals (Additional file 1). - Thus, the patterns of expression by both secretory and non- secretory genes are not driven by genetic differences among populations and the secretory ones are more likely to func- tion during insect feeding.. - To investigate whether the secretory RING proteins are injected by insects during their feeding for host manipula- tion, we selected a secretory protein, RING whose expression was upregulated in the feeding stage compared to the non-feeding stage, as the bait to test its in- teractions with plant proteins using the yeast two-hybrid assay. - 2 Comparison of secretory and non-secretory RING proteins on protein length (a), BLAST top hit E-values (b), BLAST top hit species (c), and dN/dS ratio (d). - Positions of the box plot medians are indicated by arrows, and significant differences between the secretory and non-secretory RING protein groups are indicated by asterisks (unpaired t-test p-value <. - Select genes identified as linking reproductive pathway activity to gall tissues were expressed similar as in the original study [47] in galled vs non-galled tissues (Additional file 1). - Blasts of the V. - During the genome and transcriptome curation of the gall inducing D. - 3 Phylogenies and expression profiles of secretory (left) and non-secretory RING proteins. - In contrast to the D. - vitifoliae non-secretory RING proteins, the SPRINGs are secretory, smaller (median length of 252 aa), exhibit high dN/dS values, a signature of rapid evolu- tion common among effectors, and show lineage specific duplications of genes that cluster together. - In contrast, the non-secretory RING proteins showed similar expression between the feeding adult and. - non-feeding egg stages with some even more expressed in eggs than adults, linking the non-secretory RING proteins to insect homeostasis or development. - Top: Three fragments of the bait RING protein, the full-length (RING-16-700228-FL) with the signal peptide (SP) domain removed, the RING domain (RING-16-700228-RN), and the C-terminus (RING-16-700228-C), were respectively cloned as baits for the Y2H assay. - Nevertheless, our screening revealed two candidate plant proteins, a cellulose synthase-like protein (CSLD5) and a ribosomal protein (RPS4) that bind to the RING do- main of the D. - Gene expression analyses of the ontogeny of gall development on V. - Thus, it is reasonable to hypothesize that the secretory RING proteins identified in this study are effectors of D. - Given the numer- ous effectors likely to occur in the D. - Additional study of how components of the UPS change relative to the genetic background (effector repertoire) of D. - secretory RING function, suggesting convergence in host manipulation across kingdoms. - The longest open reading frames (ORFs) for all tran- scripts of the D. - Translated protein sequences were searched against the Pfam do- main database (Pfam29.0) using the HMMSCAN pro- gram included in the HMMER software suite (version 3.1b1 [40]) for the RING clan (CL0229) which includes RING zinc finger domains and the U-box domain. - Tran- scripts were aligned to the D. - The protein sequences of the annotated gene models were subsequently retrieved, among which, the full-length or complete ones were retained for further analysis, while the incomplete ones with truncated terminus were aligned to their corresponding transcript-derived protein se- quences using BLASTP, and the terminal gaps were filled using the latter with identities ≥99%, if available. - Here, the criteria of determining full-length sequences were: 1) the presence of a conserved start codon (ATG) and a stop codon (TAA, TAG, or TGA) at the beginning and the end of the coding region, respectively. - 2) the absence of other start codon upstream of the predicted start codon within the gene sequence. - The collected RING protein genes were named based on their genomic location in the form of ‘ RING-X-Y ’ where ‘ X ’ and ‘ Y ’ indi- cated the genome scaffold number and approximate nu- cleotide position, respectively.. - Determination of putative secretory and non-secretory RING proteins. - considered as non-secretory. - Proteins with predicted transmem- brane domains were considered as non-secretory and therefore pooled together with earlier identified signal peptide-absent proteins. - vitifoliae non-secretory RINGs and SPRINGs were retrieved to search against a prefor- matted NCBI database including the non-redundant (NR) protein database and the Refseq database updated until using BLASTP (e-value ≤1e-3). - The top hit e-value and species, if available, of each query protein were used to compare the genetic conservation between the secretory and non-secretory proteins. - Estimation of the nonsy- nonymous and synonymous substitution rate ratio (dN/. - dS) was conducted in the secretory and non-secretory RING protein groups, respectively. - Although SPRINGs and RING domain containing genes were the target of the comparative analyses, we also present expres- sion profiles for egg-specific proteins and general mito- chondrial genes as positive and negative controls. - Gene ontologies for these controls were determined by search- ing annotated genes in the genome for “eggshell” and. - Phylogenetic analyses were performed on the putatively secretory and non-secretory RING finger proteins, re- spectively. - The best-fit models of protein evolution were determined using ProtTest (v3.4.2), which were FLU+G + F for the secretory proteins and WAG+G + F for the non-secretory proteins, according to Bayesian in- formation criterion. - Because the FLU protein substitu- tion matrix is not available in the phylogenetics program MRBAYES, the second best-fit model, JTT + G + F, was used instead for the phylogenetic analysis of the secretory RING proteins. - Specifically, the secretory proteins were run for 1.4 million generations to drop SDOSF below 0.05 and the non-secretory proteins were run for 2.8 million generations to drop SDOSF to 0.07, the lowest within 5 million generations of running.. - A secretory RING protein gene, RING was selected as bait to perform the yeast two-hybrid assay using the Matchmaker Gold Yeast Two-Hybrid System and the instruction supplied (Clon- tech). - plasmid construct was then transformed into Y2HGold yeast strain and the auto-activation test was conducted to ensure it did not autonomously activate the reporter genes in the absence of a prey protein. - All samples (n = 3 for each tissue type) were used in the reanalysis. - org) and enrichment was determined using a Fisher’s exact test by comparing the number of differentially expressed (DE) genes relative to controls within proteasome-related bins to the total number of DE genes for each tissue type (each gall stage, buds and flowers), and across development of the gall and leaf. - found differentially expressed or not in galled versus non- galled leaf tissue in the original dataset (Additional file 1).. - Parts of the transcriptomic resources were obtained within the 1KITE projects (Bernhard Misof, Bonn, Germany).. - The datasets supporting the conclusions of this article are included within the article (and its additional file) and in the NCBI BioProject/SRA:. - Selective proteolysis sets the tempo of the cell cycle. - Predicted effector molecules in the salivary secretome of the pea aphid (Acyrthosiphon pisum): a dual transcriptomic/proteomic approach. - A massive expansion of effector genes underlies gall-formation in the wheat pest Mayetiola destructor. - The ATL gene family from Arabidopsis thaliana and Oryza sativa comprises a large number of putative ubiquitin ligases of the RING-H2 type. - The role of the transmembrane RING finger proteins in cellular and organelle function. - Genome-wide characterization and expression profile of the grapevine ATL ubiquitin ligase family reveal biotic and abiotic stress-responsive and development-related members. - Proteasome- mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity. - Nuclear ubiquitin proteasome degradation affects WRKY45 function in the rice defense program. - Phytohormone dynamics associated with gall insects, and their potential role in the evolution of the gall-inducing habit. - Conserved and unique putative effectors expressed in the salivary glands of three related gall midge species. - De novo transcriptome assembly of the grapevine phylloxera allows identification of genes differentially expressed between leaf-and root- feeding forms. - Ontogeny of the phylloxera gall of grape leaf. - Avirulence gene mapping in the hessian fly (Mayetiola destructor) reveals a protein phosphatase 2C effector gene family. - Transcriptome analysis of the salivary glands of Nephotettix cincticeps (Uhler). - Direct roles of SPEECHLESS in the specification of stomatal self-renewing cells.
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