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Structure-guided secretome analysis of gall-forming microbes offers insights into effector diversity and evolution

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Mukhopadhyay, Soham, Javed, Muhammad Asim, Wu, Jiaxu, Perez-Lopez, Edel·2025·eLife
DOI 10.7554/elife.105185PMC PMC12503489

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Abstract

Phytopathogens secrete effector molecules to manipulate host immunity and metabolism. Recent advances in structural genomics have identified fungal effector families whose members adopt similar folds despite sequence divergence, highlighting their importance in virulence and immune evasion. To extend the scope of comparative structure-guided analysis to more evolutionarily distant phytopathogens with similar lifestyles, we used AlphaFold2 to predict the 3D structures of the secretome from selected plasmodiophorid, oomycete, and fungal gall-forming pathogens. Clustering protein folds based on structural homology revealed species-specific expansions and a low abundance of known orphan effector families. We identified novel sequence-unrelated but structurally similar (SUSS) effector clusters, rich in conserved motifs such as 'CCG' and 'RAYH'. We demonstrate that these motifs likely play a central role in maintaining the overall fold. We also identified a SUSS cluster adopting a nucleoside hydrolase-like fold conserved among various gall-forming microbes. Notably, ankyrin proteins (ANK) were significantly expanded in gall-forming plasmodiophorids, with most being highly expressed during clubroot disease, suggesting a role in pathogenicity. Subsequently, we screened ANK proteins against Arabidopsis immunity hubs using AlphaFold-Multimer and verified one of the positive results by Y2H and BiFC assays to show that the ankyrin effector PbANK1 targets host MPK3 and a zinc-binding dehydrogenase. These findings suggest a potential new mechanism in which ANK effectors target multiple host proteins involved in stress sensing, opening a novel avenue to study the role of ANK in host–pathogen interactions. Altogether, this study advances our understanding of secretome landscapes in gall-forming microbes and provides a valuable resource for broadening structural phylogenomic studies across diverse phytopathogens.

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Figure 1. Description of the pathogens included in the study, workflow overview and statistics of structure prediction. (A) Cladogram of the pathogens used in the study. The schematics represent the disease symptoms on their respective hosts, with the white areas representing galls. The secretome count indicates the number of proteins per species predicted to be secreted, and the functional annotation shows the percentage of the secretome predicted to contain a known protein domain in the Pfam, SUPERFAMILY, or Gene3D databases. (B) Flowchart of the workflow used to predict the secretome and the corresponding 3D structures. (C) Raincloud plot showing the median and density distribution of pLDDT scores of the predicted structures in each pathogen.
Figure 1. Description of the pathogens included in the study, workflow overview and statistics of structure prediction. (A) Cladogram of the pathogens used in the study. The schematics represent the disease symptoms on their respective hosts, with the white areas representing galls. The secretome count indicates the number of proteins per species predicted to be secreted, and the functional annotation shows the percentage of the secretome predicted to contain a known protein domain in the Pfam, SUPERFAMILY, or Gene3D databases. (B) Flowchart of the workflow used to predict the secretome and the corresponding 3D structures. (C) Raincloud plot showing the median and density distribution of pLDDT scores of the predicted structures in each pathogen.CC BY · PubMed Central
Figure 2. Visualization of dominant protein folds present in each pathogen. (Top) Network plot of structurally similar secretome clusters with at least 15 members. Not all 255 clusters are shown to reduce complexity. Each node represents a single protein, and an edge between two nodes represents structural similarity (TMScore >0.5). (Bottom) Representative structure of the dominant fold in each pathogen. Since Ankyrin repeats are common in both P. brassicae and S. subterranea, they are represented only once.
Figure 2. Visualization of dominant protein folds present in each pathogen. (Top) Network plot of structurally similar secretome clusters with at least 15 members. Not all 255 clusters are shown to reduce complexity. Each node represents a single protein, and an edge between two nodes represents structural similarity (TMScore >0.5). (Bottom) Representative structure of the dominant fold in each pathogen. Since Ankyrin repeats are common in both P. brassicae and S. subterranea, they are represented only once.CC BY · PubMed Central
Figure 2—figure supplement 1. The structures of Beta vulgari’s GTPases, KP6 fold, RALPH-like fold, and ToxA fold share structural similarity with candidate effectors found in P. betae, U. maydis, and T. deformans, respectively. The number of clusters where proteins share structural similarities with these folds is provided.
Figure 2—figure supplement 1. The structures of Beta vulgari’s GTPases, KP6 fold, RALPH-like fold, and ToxA fold share structural similarity with candidate effectors found in P. betae, U. maydis, and T. deformans, respectively. The number of clusters where proteins share structural similarities with these folds is provided.CC BY · PubMed Central
Figure 3. Sequence and structural similarity among HopQ1 homologs in U. maydis, A. candida, and P. brassicae. (A) Network plot showing the structural similarity between the members of cluster 21. Edges denote structural similarity (TMScore >0.5). (B) Pairwise sequence identity between selected HopQ1 structural homologs from plasmodiophorids, oomycetes, and fungi, illustrating sequence dissimilarity between some proteins despite structural homology. (C) Gene expression values (log2 TPM) of two highly induced P. brassicae genes at 16 and 26 dpi. (D) 3D structure of the mature protein sequences, assuming a HopQ1-like fold.
Figure 3. Sequence and structural similarity among HopQ1 homologs in U. maydis, A. candida, and P. brassicae. (A) Network plot showing the structural similarity between the members of cluster 21. Edges denote structural similarity (TMScore >0.5). (B) Pairwise sequence identity between selected HopQ1 structural homologs from plasmodiophorids, oomycetes, and fungi, illustrating sequence dissimilarity between some proteins despite structural homology. (C) Gene expression values (log2 TPM) of two highly induced P. brassicae genes at 16 and 26 dpi. (D) 3D structure of the mature protein sequences, assuming a HopQ1-like fold.CC BY · PubMed Central
Figure 4. Sequence and structural similarity among Mig1 homologs in Ustilago maydis. (A) Similarity matrix showing the pairwise sequence identity (%) between Mig1 cluster members. (B) Similarity matrix showing the pairwise structural homology scores (TMScore) between Mig1 cluster members. (C) Superimposition of two Mig1 homologues, illustrating structural similarity despite extreme sequence divergence. (D) Differential gene expression patterns of two Mig1 tandem duplicates. (E) Multiple alignment of protein sequences, highlighting the conservation of cysteine residues (marked in yellow). (F) Visualization of the conserved cysteine residues forming disulfide bridges.
Figure 4. Sequence and structural similarity among Mig1 homologs in Ustilago maydis. (A) Similarity matrix showing the pairwise sequence identity (%) between Mig1 cluster members. (B) Similarity matrix showing the pairwise structural homology scores (TMScore) between Mig1 cluster members. (C) Superimposition of two Mig1 homologues, illustrating structural similarity despite extreme sequence divergence. (D) Differential gene expression patterns of two Mig1 tandem duplicates. (E) Multiple alignment of protein sequences, highlighting the conservation of cysteine residues (marked in yellow). (F) Visualization of the conserved cysteine residues forming disulfide bridges.CC BY · PubMed Central
Figure 5. SUSS effector families are enriched in common motifs. (A) Network plots demonstrating that the two primary effector families in A. candida and S. endobioticum can only be grouped together when structural data is incorporated into the sequence-based clustering. The plots also indicate which sequence-based clusters contain the known effectors from these groups. (B) ‘RAYH’ and ‘CCG’ motif patterns identified by MEME scan. (C) Disulfide bridges in the ‘CCG’ motif, likely playing a pivotal role in structural maintenance, are highlighted in the virulence factor CCG30. A zoomed-in view of the ‘CCG module’ shows the four conserved cysteine residues forming disulfide bridge. (D) The ‘RAYH’ motif, occupying the central position in the core alpha-helix bundle, is highlighted in six sequence-based subclusters within the AvrSen1-like cluster in S. endobioticum.
Figure 5. SUSS effector families are enriched in common motifs. (A) Network plots demonstrating that the two primary effector families in A. candida and S. endobioticum can only be grouped together when structural data is incorporated into the sequence-based clustering. The plots also indicate which sequence-based clusters contain the known effectors from these groups. (B) ‘RAYH’ and ‘CCG’ motif patterns identified by MEME scan. (C) Disulfide bridges in the ‘CCG’ motif, likely playing a pivotal role in structural maintenance, are highlighted in the virulence factor CCG30. A zoomed-in view of the ‘CCG module’ shows the four conserved cysteine residues forming disulfide bridge. (D) The ‘RAYH’ motif, occupying the central position in the core alpha-helix bundle, is highlighted in six sequence-based subclusters within the AvrSen1-like cluster in S. endobioticum.CC BY · PubMed Central
Figure 5—figure supplement 1. Location of CCG motifs identified by MEME scan.
Figure 5—figure supplement 1. Location of CCG motifs identified by MEME scan.CC BY · PubMed Central
Figure 5—figure supplement 2. Location of RAYH motifs identified by MEME scan.
Figure 5—figure supplement 2. Location of RAYH motifs identified by MEME scan.CC BY · PubMed Central
Figure 5—figure supplement 3. Analysis evidencing purifying selection in the CCG effector family. Plots have been downloaded from the Datamonkey server, which hosts the FEL package. The indicated codons are numbered according to the codon-aligned nucleotide alignment file, where positions with 50% gaps have been trimmed.
Figure 5—figure supplement 3. Analysis evidencing purifying selection in the CCG effector family. Plots have been downloaded from the Datamonkey server, which hosts the FEL package. The indicated codons are numbered according to the codon-aligned nucleotide alignment file, where positions with 50% gaps have been trimmed.CC BY · PubMed Central
Figure 5—figure supplement 4. Multiple alignment of representative members of CCG sequence-based clusters. Conserved residues (>70% similar by biochemical properties in ESPript3.0) are highlighted.
Figure 5—figure supplement 4. Multiple alignment of representative members of CCG sequence-based clusters. Conserved residues (>70% similar by biochemical properties in ESPript3.0) are highlighted.CC BY · PubMed Central
Figure 5—figure supplement 5. Multiple alignment of representative members of AvrSen1-like sequence-based clusters. Conserved residues (>70% similar by biochemical properties in ESPript3.0) are highlighted.
Figure 5—figure supplement 5. Multiple alignment of representative members of AvrSen1-like sequence-based clusters. Conserved residues (>70% similar by biochemical properties in ESPript3.0) are highlighted.CC BY · PubMed Central
Figure 6. Diversity, structural features, and host immune targets of ankyrin repeats in Plasmodiophorids. (A) Frequency of repeat-containing proteins in P. brassicae and S. subterranea. (B) Network plot showing structural homology within P. brassicae Ankyrin repeats, also highlighting the Ankyrin repeats with SKP1/BTB/POZ superfamily domains. (C) Alignment of Ankyrin motifs from P. brassicae and S. subterranea. (D) Visualization of conserved hydrophobic residues in a single Ankyrin repeat module. (E) Number of Ankyrin repeat proteins predicted to target Arabidopsis immune proteins. (F) AlphaFold Multimer predicted complex of MPK3 and PbANK1 (PBTT_00818), highlighting the predicted aligned errors of surface contacts under 4 Ångströms.
Figure 6. Diversity, structural features, and host immune targets of ankyrin repeats in Plasmodiophorids. (A) Frequency of repeat-containing proteins in P. brassicae and S. subterranea. (B) Network plot showing structural homology within P. brassicae Ankyrin repeats, also highlighting the Ankyrin repeats with SKP1/BTB/POZ superfamily domains. (C) Alignment of Ankyrin motifs from P. brassicae and S. subterranea. (D) Visualization of conserved hydrophobic residues in a single Ankyrin repeat module. (E) Number of Ankyrin repeat proteins predicted to target Arabidopsis immune proteins. (F) AlphaFold Multimer predicted complex of MPK3 and PbANK1 (PBTT_00818), highlighting the predicted aligned errors of surface contacts under 4 Ångströms.CC BY · PubMed Central
Figure 7. Validation of PbANK1-MPK3 and PbANK1-GroES-like interactions through Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC). (A) 1-by-1 Y2H assay results evaluating the interaction PbANK1-MPK3 (AT3G45640) predicted through AlphaFold-Multimer. N=3. (B) 1-by-1 Y2H assay results evaluating the interaction PbANK1-GroES-like (AT3G56460) predicted through a Y2H screening of an Arabidopsis seedling library. N=3. (C) BiFC assay results evaluating the interaction PbANK1-MPK3. N=3, presented in Figure 7—figure supplement 1. Bar = 50 μm. (D) BiFC assay results evaluating the interaction PbANK1- GroES-like. N=3, presented in Figure 7—figure supplement 2. Bar = 50 μm.
Figure 7. Validation of PbANK1-MPK3 and PbANK1-GroES-like interactions through Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC). (A) 1-by-1 Y2H assay results evaluating the interaction PbANK1-MPK3 (AT3G45640) predicted through AlphaFold-Multimer. N=3. (B) 1-by-1 Y2H assay results evaluating the interaction PbANK1-GroES-like (AT3G56460) predicted through a Y2H screening of an Arabidopsis seedling library. N=3. (C) BiFC assay results evaluating the interaction PbANK1-MPK3. N=3, presented in Figure 7—figure supplement 1. Bar = 50 μm. (D) BiFC assay results evaluating the interaction PbANK1- GroES-like. N=3, presented in Figure 7—figure supplement 2. Bar = 50 μm.CC BY · PubMed Central
Figure 7—figure supplement 1. Extended BiFC assay results evaluating the interaction PbANK1-MPK3 presented in Figure 7c including the three replicates, empty vectors, and positive controls.
Figure 7—figure supplement 1. Extended BiFC assay results evaluating the interaction PbANK1-MPK3 presented in Figure 7c including the three replicates, empty vectors, and positive controls.CC BY · PubMed Central
Figure 7—figure supplement 2. Extended BiFC assay results evaluating the interaction PbANK1-GroES-like presented in Figure 7d including the three replicates, empty vectors, and positive controls.
Figure 7—figure supplement 2. Extended BiFC assay results evaluating the interaction PbANK1-GroES-like presented in Figure 7d including the three replicates, empty vectors, and positive controls.CC BY · PubMed Central

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Metadata

Authors
Mukhopadhyay, S., Javed, M. A., Wu, J., & Perez-Lopez, E.
Affiliations
  • Université Laval (CA)
  • Départment de Phytologie, Faculté des sciences de l’agriculture et de l’alimentation, Université Laval
Corresponding author
Mukhopadhyay, Soham
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2025-10-07
Volume
14
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.105185
PMC
PMC12503489
OpenAlex
W4407840168
Cited by
5
References
4
Citations by year
2026: 4 · 2025: 1
Type
posted content
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Canola Council of Canada (2021.4)
  • Natural Sciences and Engineering Research Council of Canada (RGPIN-2021-02518)
  • Western Grain Research Foundation
  • Manitoba Canola Growers Association
  • Alberta Canola Producers Commission
  • Fonds de recherche du Québec (Doctoral scholarships)
Topics
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Disease Resistance and Genetics
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant-Microbe Interactions and Immunity
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Pathogens and Resistance
Keywords
secretome, effector proteins, oomycete, plant pathogenic fungi, structural genomics, clubroot disease, MPK3, host-pathogen interaction, effector evolution
MeSH terms
Secretome, Fungal Proteins, Fungi, Oomycetes, Plant Diseases, Plant Tumors, Arabidopsis, Evolution, Molecular

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