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At least 19 recordsLinked to original sources

PubMed2025

In silico prediction method for plant Nucleotide-binding leucine-rich repeat- and pathogen effector interactions.

Plant Nucleotide-binding leucine-rich repeat (NLR) proteins play a crucial role in effector recognition and activation of Effector triggered immunity following pathogen infection. Genome sequencing advancements have led to the identification of a myriad of NLRs in numerous agriculturally important plant species. However, deciphering which NLRs recognize specific pathogen effectors remains challenging. Predicting NLR-effector interactions in silico will provide a more targeted approach for experimental validation, critical for elucidating function, and advancing our understanding of NLR-triggered immunity. In this study, NLR-effector protein complex structures were predicted using AlphaFold2-Multimer for all experimentally validated NLR-effector interactions reported in literature. Binding affinities- and energies were predicted using 97 machine learning models from Area-Affinity. We show that AlphaFold2-Multimer predicted structures have acceptable accuracy and can be used to investigate NLR-effector interactions in silico. Binding affinities for 58 NLR-effector complexes ranged between -8.5 and -10.6 log(K), and binding energies between -11.8 and -14.4 kcal/mol-1, depending on the Area-Affinity model used. For 2427 "forced" NLR-effector complexes, these estimates showed larger variability, enabling identification of novel NLR-effector interactions with 99% accuracy using an Ensemble machine learning model. The narrow range of binding energies- and affinities for "true" interactions suggest a specific change in Gibbs free energy, and thus conformational change, is required for NLR activation. This is the first study to provide a method for predicting NLR-effector interactions, applicable to all pathosystems. Finally, the NLR-Effector Interaction Classification (NEIC) resource can streamline research efforts by identifying NLRs important for plant-pathogen resistance, advancing our understanding of plant immunity.

Plant Proteins↗

PubMed2025

Transposable elements create distinct genomic niches for effector evolution among Magnaporthe oryzae lineages.

BACKGROUND: Plant-pathogen interactions are characterized by evolutionary arms races. At the molecular level, fungal effectors can target important plant functions, while plants evolve to improve effector recognition. Rapid evolution in genes encoding effectors can be facilitated by transposable elements (TEs). In Magnaporthe oryzae, the causal agent of blast disease in several cereals and grasses, TEs play important roles in chromosomal evolution as well as the gain or loss of effector genes in host specialized lineages. However, a global understanding of TE dynamics driving effector evolution at population scale and across lineages is lacking. RESULTS: Here, we focus on 16 AVR effector loci assessed across a global sampling of 11 reference genomes and 447 newly generated draft genome assemblies from publicly available short-read sequencing data across all major M. oryzae lineages and outgroups. We classified each effector based on evidence for duplication, deletion and translocation processes among lineages. Next, we determined AVR gain and loss dynamics across lineages allowing for a broad categorization of effector dynamics. Each AVR was integrated in a distinct genomic niche determined by the TE activity profile contributing to the diversification at the locus. We quantified TE contributions to effector niches and found that TE identity helped diversify AVR loci. We used the large genomic dataset to recapitulate the evolution of the rice blast AVR1-CO39 locus. CONCLUSIONS: Taken together, our work demonstrates how TE dynamics are an integral component of M. oryzae effector evolution, likely facilitating escape from host recognition. In-depth tracking of effector loci is a valuable tool to predict the durability of host resistance.

Ascomycota↗

PubMed2026

Systematic discovery of pathogen effector functions across human pathogens and pathways.

Pathogens deploy effector proteins to exploit host cell biology, and most effector open reading frames (ORFs) are rapidly evolving and lack functional annotation. We developed the effector ORFeome (eORFeome), a scalable functional genomics platform encompassing 3,835 effector ORFs from diverse viruses, bacteria, and parasites. High-throughput barcoded screens across nuclear factor κB (NF-κB), apoptosis, p53, cGAS-STING, and major histocompatibility complex class I (MHC class I) pathways revealed novel pathway-modulating functions for hundreds of uncharacterized eORFs, unexpected activities of known effectors, and distinct pathway-specific functions encoded by single ORFs. Illustrating the power of this approach, we identified HHV6A U14 as a p53 antagonist, HHV7 U21 as a dual-function STING antagonist and MHC-I antigen display inhibitor, and adenoviral 13.6K/i-leader protein as a de novo-evolved TAP inhibitor that suppresses MHC-I display. These results establish a general framework for systematic effector annotation, uncover new mechanisms of host-pathogen interaction across kingdoms, and highlight pathogen effectors as a versatile toolkit for rewiring and probing human cellular pathways.

Humans↗

PubMed2026

Systematic Identification and Functional Characterisation of Colletotrichum fructicola Effectors During Camellia oleifera Colonisation.

Camellia oleifera is an important woody oil crop in southern China, but its production is severely threatened by anthracnose caused by Colletotrichum fructicola. C. fructicola deploys secreted effector proteins to establish infection. However, systematic identification and functional characterisation of C. fructicola effector genes upregulated during infection remains largely unexplored. Here, we integrated genome-wide secretome prediction with RNA-seq data from C. oleifera leaves inoculated with C. fructicola to identify candidate effectors induced during infection, followed by functional screening, targeted gene deletion, complementation and pathogenicity assays. Five novel effectors required for C. fructicola full virulence were identified, all of which suppressed Bax-induced cell death in Nicotiana benthamiana. Targeted deletion of the corresponding genes in C. fructicola reduced lesion areas by 47%-78% on C. oleifera leaves and by up to 67% on apple fruits, whereas complementation restored their virulence to wild-type levels. Transcriptomic profiling of infected postharvest C. oleifera fruits identified differentially expressed genes enriched in GO terms related to copper ion response, as well as in KEGG pathways associated with phenylpropanoid biosynthesis, taurine and hypotaurine metabolism, and plant-pathogen interactions. In addition, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities, as well as malondialdehyde content, were altered in C. oleifera leaves inoculated with the effector-deletion mutants compared with those infected with the wild-type strain, suggesting that these effectors may contribute to the modulation of host oxidative stress responses. Taken together, our findings provide genetic and physiological evidence that these effectors contribute to C. fructicola virulence, offering potential targets for anthracnose control.

Colletotrichum↗

PubMed2026

Decoding Primary Open-Angle Glaucoma: A Multi-Omics Approach to Identify Druggable Effector Genes.

PURPOSE: Genomewide association studies (GWAS) have identified numerous primary open angle glaucoma (POAG) risk loci, yet most reside in non-coding regions with unclear function. Mapping these loci to effector genes can elucidate disease mechanisms, identify functionally conserved variants, improve cross-ancestry risk prediction by reducing population-specific noise, and uncover shared therapeutic targets. METHODS: Here, we integrate European POAG GWAS with six types of multi-omics molecular Quantitative Trait Locis (xQTLs) using multi-trait colocalization to identify candidate effector variants and evaluate their cross-population relevance using genetic risk score (GRS) analysis, and their therapeutic potential through drug target prioritization. RESULTS: We identified 25 POAG effector variants colocalized with at least one xQTLs. In non-European populations, effector variants showed stronger effect size correlations with Europeans than non-colocalized variants (Pearson r2 = African 0.85 vs. 0.71; East Asian 0.81 vs. 0.69; and Latin American 0.91 vs. 0.75). Effector variants also had smaller allele frequency variations across populations (average interquartile range [IQR] = 0.15 vs. 0.20). The genetic risk score based on effector variants performed comparably to the genome-wide significant single-nucleotide polymorphism (SNP)-based GRS in non-European populations. Drug prioritization identified zinc, copper, sunitinib, probucol, and astemizole as potential common therapeutic agents for POAG and its subtypes. CONCLUSIONS: Our findings offer deeper insight into the molecular mechanisms underlying glaucoma and effector variants for developing more robust GRS models and broadly effective therapeutic strategies for POAG.

Humans↗

PubMed2026

Integrated Functional Characterization of Hemileia vastatrix Effector Candidates Reveals Coordinated Immune Suppression, Sequential Deployment and Compartment-Specific Targeting.

Coffee leaf rust, caused by the obligate biotrophic fungus Hemileia vastatrix, remains the most destructive disease of coffee worldwide. Although genomic and transcriptomic studies have identified a large number of candidate effectors, experimental evidence supporting their biological roles during infection remains limited. Here, we integrated functional assays, temporal expression profiling during coffee infection and subcellular localization analyses to investigate the biological properties of 44 H. vastatrix effector candidates (HvECs). Using the Pseudomonas fluorescens EtHAn effector delivery system in Nicotiana benthamiana, 15 HvECs consistently suppressed pattern-triggered immunity (PTI), indicating that immune suppression is a widespread property among the H. vastatrix effector repertoire, as assessed in this heterologous system. Five HvECs also attenuated AvrB-triggered effector-triggered immunity (ETI), and three suppressed both PTI and ETI, suggesting that a subset of HvECs targets conserved regulatory nodes shared by these interconnected immune pathways. Temporal expression profiling revealed sequential deployment of HvECs throughout infection, with distinct subsets predominating during pre-biotrophic development, host penetration or biotrophic colonization, consistent with stage-specific functions during fungal pathogenesis. Subcellular localization analyses further showed that HvECs preferentially accumulated in the nucleus or chloroplasts, compartments known as central hubs of plant immune regulation. This study provides the most comprehensive functional characterization of H. vastatrix effector candidates to date, establishes a biologically informed framework for prioritizing candidates for future identification of avirulence determinants recognized by SH resistance genes, and advances our understanding of how the coffee rust fungus orchestrates immune suppression across time and cellular space during pathogenesis.

Nicotiana↗

PubMed2025

Diversification, loss, and virulence gains of the major effector AvrStb6 during continental spread of the wheat pathogen Zymoseptoria tritici.

Interactions between plant pathogens and their hosts are highly dynamic and mainly driven by pathogen effectors and plant receptors. Host-pathogen co-evolution can cause rapid diversification or loss of pathogen genes encoding host-exposed proteins. The molecular mechanisms that underpin such sequence dynamics remains poorly investigated at the scale of entire pathogen species. Here, we focus on AvrStb6, a major effector of the global wheat pathogen Zymoseptoria tritici, evolving in response to the cognate receptor Stb6, a resistance widely deployed in wheat. We comprehensively captured effector gene evolution by analyzing a global thousand-genome panel using reference-free sequence analyses. We found that AvrStb6 has diversified into 59 protein isoforms with a strong association to the pathogen spreading to new continents. Across Europe, we found the strongest differentiation of the effector consistent with high rates of Stb6 deployment. The AvrStb6 locus showed also a remarkable diversification in transposable element content with specific expansion patterns across the globe. We detected AvrStb6 gene losses and evidence for transposable element-mediated disruptions. We used virulence datasets of genome-wide association mapping studies to predict virulence changes across the global panel. Genomic predictions suggested marked increases in virulence on Stb6 cultivars concomitant with the spread of the pathogen to Europe and the subsequent spread to further continents. Finally, we genotyped French bread wheat cultivars for Stb6 and monitored resistant cultivar deployment concomitant with AvrStb6 evolution. Taken together, our data provides a comprehensive view of how a rapidly diversifying effector locus can undergo large-scale sequence changes concomitant with gains in virulence on resistant cultivars. The analyses highlight also the need for large-scale pathogen sequencing panels to assess the durability of resistance genes and improve the sustainability of deployment strategies.

Ascomycota↗

PubMed2025

Annotation of RxLR Effectors in Oomycete Genomes.

Pathogens have evolved effector proteins to suppress host immunity and facilitate plant infections. RxLR effectors are small, secreted effector proteins with conserved RxLR and dEER amino acid motifs at the N terminus and highly variable C termini and are commonly found in oomycete species. We provide computational approaches to annotate RxLR candidate effector genes in a genome assembly in FASTA format with an available GFF file. Hidden Markov Modeling (HHM) is used in combination with regular expressions to search for RxLR and EER amino acid patterns.

Oomycetes↗

PubMed2025

Multiple effectors trigger non-host resistance in Solanum americanum against Pseudomonas syringae.

Wild plant species are threatened by diverse pathogens, but disease symptoms are rarely observed in nature. This suggests that wild plants harbor valuable sources of resistance. In this study, we show that the model bacterial pathogen Pseudomonas syringae pv. tomato (Pto) DC3000 triggered defense responses in all tested accessions of a wild Solanaceae species, Solanum americanum. Pto DC3000-triggered immunity in S. americanum required a type III secretion system. We show that seven Pto DC3000 effectors (AvrPto, HopAD1, HopAM1, HopC1, HopAA1-1, HopM1, and AvrE1) triggered hypersensitive responses (HR) in S. americanum accession SP2273. Significantly, sequential deletion of the HR-triggering effectors from Pto DC3000 resulted in enhanced virulence in S. americanum. However, the well-conserved effectors, HopM1 and AvrE1, were indispensable for virulence. We conclude that the immunity triggered by multiple effectors contributes to nonhost resistance in S. americanum against P. syringae. We propose that the identification of the corresponding disease resistance genes for HopM1 and AvrE1 in S. americanum would accelerate the development of durable immunity to P. syringae pathogens in Solanaceae crops.

Disease Resistance↗

PubMed2026

Sequential Immune Activation of Effector T Cells as Biomarkers of Response to Durvalumab in Patients with Locally Advanced NSCLC.

PURPOSE: Durvalumab therapy following concurrent chemoradiotherapy (cCRT) improves progression-free survival (PFS) in patients with unresectable locally advanced non-small cell lung cancer. In this prospective observational study, we evaluated the changes in peripheral blood immune cell counts to elucidate the immunologic mechanisms underlying cCRT and durvalumab therapy. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cell (PBMC) samples were collected at four time points: before cCRT, after cCRT, at the start of durvalumab, and 8 weeks after the start of durvalumab, and analyzed by multicolor flow cytometry. RESULTS: Of the 149 enrolled patients, 115 received durvalumab consolidation therapy after cCRT. The median PFS in the overall population was 24.2 months, and the 3-year PFS rate was 38.9%. PBMC analysis showed an increased effector fraction of CD4+ T cells before and after cCRT but no change in CD8+ T cells. Following durvalumab therapy, the effector fraction ratio of CD8+ T cells (CD62Llow CD8+ T cells) increased and positively correlated with increased CD62Llow CD4+ T cells during cCRT. Patients whose proportion of CD62Llow CD4+ T cells exceeded the threshold for cCRT had better PFS than those below the threshold. Patients whose CD62Llow CD8+ T-cell proportion exceeded the threshold after durvalumab therapy showed prolonged PFS compared with those below the threshold. CONCLUSIONS: cCRT promotes an increase in effector CD4+ T cells, and the subsequent increase in CD8+ T cells following durvalumab therapy prolongs PFS. Peripheral blood effector-type CD4+ and CD8+ T cells are potential biomarkers for evaluating the immune status of patients and predicting treatment efficacy.

Humans↗

PubMed2026

From Sequential Gland Replacement to Recurrent Gland Coordination: A Comparative Framework for Subventral and Dorsal Oesophageal Gland Effectors Across Plant-Parasitic Nematode Lifestyles.

Plant-parasitic nematodes manipulate host tissues through stylet-secreted gene products synthesised principally in two subventral and one dorsal oesophageal gland. Earlier reviews have catalogued effector repertoires, described feeding-site formation, and explained how individual effectors modify host defence, development, and metabolism. However, the temporal coordination of the gland cells themselves has not been comparatively synthesised across parasitic lifestyles. This review therefore advances a gland-centred, lifestyle-dependent framework. In sedentary endoparasites, available evidence supports a pronounced developmental transition: subventral gland products dominate penetration and migration, whereas dorsal gland products become increasingly important during feeding-site initiation and maintenance. Migratory endoparasites repeatedly penetrate, migrate, and feed without establishing permanent feeding cells; their gland activity is consequently predicted to be recurrent and overlapping rather than a one-way replacement. Ectoparasites likewise require behaviour-dependent coordination during repeated probing and external feeding, although direct gland localisation evidence remains limited. We integrate gland origin, secretion chemistry, infection stage, and parasitic behaviour across root-knot, cyst, citrus, false root-knot, lesion, burrowing, and ectoparasitic nematodes. The synthesis distinguishes experimentally demonstrated gland localisation from evidence-weighted inference and formulates testable predictions for comparative gland transcriptomics, spatial expression, and functional silencing. This framework also identifies gland activation, secretion, and stage-critical products as targets for RNA interference, genome editing, resistance breeding, and sustainable nematode management. The principal novelty is therefore not another catalogue of nematode effectors, but a comparative model explaining when and why subventral and dorsal glands exchange, retain, or alternate their functions across contrasting parasitic lifestyles.

dorsal gland↗

PubMed2026

Mapping antibody sequences and effector functions across spatial niches.

Antibodies are fundamental to human health but can also drive pathology. Each antibody has a molecular specificity, encoded by their clonally heritable B cell receptor (BCR). Recent advances in spatial transcriptomics coupled with repertoire sequencing have enabled capturing antibody-secreting cells (ASCs) and their clonal BCR within their tissue microenvironment. However, our understanding of antibody production niches remains limited. Furthermore, where antibodies are produced can be distinct from where antibodies exert their effector function. Here, we propose a conceptual spatial framework to distinguish between 'antibody production niches', defined by the ASC, BCR, and niche composition, versus 'antibody functional niches', composed of the antibody, antigen, and effector landscape. We then examine the possibilities and challenges to map and link antibody-encoding sequences and antibody effector functions using current and emerging technologies. Combined, we argue that integrating spatial sequence data with the antibody functional context is essential to decode the architecture of antibody-mediated immunity.

Humans↗

PubMed2025

Effectidor II: a pan-genomic AI-based algorithm for the prediction of type III secretion system effectors.

MOTIVATION: Type III secretion systems are used by many Gram-negative bacteria to inject type 3 effectors (T3Es) directly into eukaryotic cells, promoting disease or provoking immune response. Because of these opposing evolutionary forces, T3E repertoires often vary within taxonomic groups. Identifying the full effector gene repertoire in genomes of related individuals is crucial for determining core and specialized effectors, understanding the disease dynamics, and developing appropriate management strategies against pathogens. It can also help uncover novel T3Es that have recently emerged in a population. Our previously published Effectidor web server successfully addressed the challenge of identifying T3Es in a single bacterial genome. Here, we enriched the web server with various novel capabilities, including the identification of T3Es from multiple genome sequences simultaneously. RESULTS: We present Effectidor II, a web server that relies on machine learning to predict T3E-encoding genes within bacterial pan-genomes. We demonstrate the benefit of learning based on features extracted from the entire sequences comprising the pan-genome and report a novel T3E discovered by it in Xanthomonas euroxanthea. AVAILABILITY AND IMPLEMENTATION: Effectidor II is available at: https://effectidor.tau.ac.il and the source code is available at: https://github.com/naamawagner/Effectidor. A stand-alone version of Effectidor II is available at: https://github.com/naamawagner/Effectidor/tree/StandAlone. The source code for the standalone version and the data used in this work are also provided in https://doi.org/10.5281/zenodo.15081636.

Type III Secretion Systems↗

PubMed2024

A gene with a thousand alleles: The hyper-variable effectors of plant-parasitic nematodes.

Pathogens are engaged in a fierce evolutionary arms race with their host. The genes at the forefront of the engagement between kingdoms are often part of diverse and highly mutable gene families. Even in this context, we discovered unprecedented variation in the hyper-variable (HYP) effectors of plant-parasitic nematodes. HYP effectors are single-gene loci that potentially harbor thousands of alleles. Alleles vary in the organization, as well as the number, of motifs within a central hyper-variable domain (HVD). We dramatically expand the HYP repertoire of two plant-parasitic nematodes and define distinct species-specific "rules" underlying the apparently flawless genetic rearrangements. Finally, by analyzing the HYPs in 68 individual nematodes, we unexpectedly found that despite the huge number of alleles, most individuals are germline homozygous. These data support a mechanism of programmed genetic variation, termed HVD editing, where alterations are locus specific, strictly governed by rules, and theoretically produce thousands of variants without errors.

Animals↗

PubMed2026

Phytoplasma-plant interactions: effector-mediated host reprogramming, hormonal crosstalk, metabolic alterations and plant-mediated vector manipulation.

Phytoplasmas are wall-less, phloem-restricted bacterial pathogens that infect over 1,000 plant species, causing substantial losses in agriculture, horticulture, and forestry worldwide. Despite their reduced genomes and limited metabolic autonomy, these obligate parasites colonize diverse hosts through secreted effector proteins that extensively reprogram plant development, metabolism, immune signalling, and vector interactions. Advances in genomics, transcriptomics, proteomics, metabolomics, and functional studies have substantially clarified the molecular basis of phytoplasma pathogenicity and symptom development. This review synthesizes current understanding of phytoplasma-plant interactions, covering phytoplasma biology, genome evolution, and the infection cycle across plant and insect vector hosts. We examine the molecular functions of key effectors, SAP11, SAP54/PHYL1, SAP05, TENGU, SWP1, and recently identified virulence factors, focusing on how they target host transcription factors, phytohormone networks, protein degradation pathways, and immune responses to promote colonization and disease progression. We further discuss how phytoplasma infection disrupts phytohormone signalling, primary and secondary metabolism, and developmental programs to produce characteristic disease symptoms, with particular attention to pathogen-induced changes in host volatiles and nutritional quality that alter vector behaviour and enhance transmission. Finally, we summarize insights from multi-omics studies and emerging management strategies, including CRISPR-based genome editing, RNAi, rapid molecular diagnostics, resistant cultivars, microbiome-based approaches, and sustainable vector control, and highlight key knowledge gaps and priorities for developing effective, environmentally sustainable phytoplasma disease management.

Phytoplasma↗

PubMed2026

Aphid symbiotic virus engineered for in vivo expression of insecticidal effectors.

Microbial pesticides are eco-friendly alternatives to chemical pesticides. However, few viral pesticides have been developed. Insects harbor diverse symbiotic viruses, which have the potential to be engineered for translational applications in pest control. Here, we engineered Acyrthosiphon pisum virus (APV), a symbiotic virus of the pea aphid, to deliver anti-aphid effectors using reverse genetics technology. A cytomegalovirus (CMV) promoter-driven APV infectious clone was successfully rescued in pea aphids with the assistance of nanocarrier star polymer (SPc). Based on this infectious clone, the protein coding sequence of chymotrypsin inhibitor variant 8 (Chy8) and the double-stranded RNA sequence targeting the aphid clip-domain serine protease (SPLP) were separately assembled into the APV genome to generate APV-Chy8 and APV-dsSPLP infectious clones, respectively. The recombinant APV clones reduced aphid relative survival rates by 34% and 17% by microinjection, respectively. To enhance the transcriptional efficiency, the APV-Chy8 and APV-dsSPLP clones were transcribed in vitro using the T7 promoter. The in vitro-synthesized APV-Chy8 and APV-dsSPLP clones reduced aphid relative survival rates by 48% and 45% by microinjection, respectively. These results demonstrate that engineered APV can deliver cargos and reduce aphid survival under injection-based experimental conditions, highlighting the potential of symbiotic virus-based vectors for delivering insecticidal effectors.

Animals↗

PubMed2026

Effector loss and gain drives host range at a fitness cost.

Epidemic preparedness depends on tracking microbial evolution that drives shifts in ecological behaviors such as disease emergence. However, the genetic constraints mediating microbial emergence for generalist and specialist behaviors remain poorly described. Here, we addressed this question by combining comparative and functional genomics with phylogeny-based evolutionary analyses of the cereal pathogen Xanthomonas translucens. We show that a generalist X. translucens subgroup arose from a specialist ancestor, and the loss of a single effector gene, xopAL1, contributed to the generalist host expansion by promoting host jump from barley to wheat. Deleting barley-specialist X. translucens xopAL1 recapitulated the host jump to wheat and demonstrates risk across each globally distributed genetic lineage. However, this niche expansion via XopAL1 loss incurs a significant fitness cost to colonize barley. Moreover, the specialist lineage gained an additional effector gene, xopAJ, which enhanced virulence on barley while restricting oat infection, thereby reinforcing niche specialization. We further conducted transcriptomic analysis of wheat and determined that XopAL1 triggers a defense response that involves the reduction of photosynthetic processes. Our work provides an experimentally validated evolutionary framework to understand mechanisms of intergenera host jump. Overall, we demonstrate that single events of gene loss and gain shape ecological behaviors by creating a dynamic trade-off between niche breadth and specialization.

Triticum↗

PubMed2026

Rapidly evolving aphid gall effector proteins exhibit saposin-like folds.

Many insects manipulate plants by injecting effector proteins. In one extreme example of this molecular "hijacking," Hormaphis cornu aphids inject bicycle proteins into Hamamelis virginiana, contributing to the development of novel organs called galls. Bicycle proteins share no amino acid sequence similarity with proteins of known function. Here, we report the crystal structures of two divergent bicycle proteins. Both proteins contain saposin-like folds: one with multiple disulfide bonds exhibits a swapped domain topology; the other has no disulfide bonds and possesses two distinct, tandem domains. To explore the structural evolution of bicycle proteins, we attempted to predict bicycle protein structures with Alphafold2 (AF2) and other deep learning programs. While AF2 did not recover the two experimental structures using existing databases, it succeeded when provided with multiple sequence alignments (MSAs) of protein sequences from newly sequenced closely related species. Using this approach, we generated 2,400 high-confidence bicycle protein predictions from seven aphid species. While all aphid bicycle proteins contain predicted saposin-like folds, they display a vast diversity of structural and physicochemical properties. While this diversity thwarts prediction of conserved functions encoded in structure, it suggests that bicycle proteins have evolved to target diverse plant processes and/or to evade plant immune surveillance. Our extension of AF2 with custom MSAs of proteins from closely related species provides a generalizable, powerful approach for predicting structures of rapidly evolving protein families.

Animals↗