Gold-Tagged Antibodies Expose Sur7 Marker on Fungal Pathogen Vesicles

Using an antibody raised against the protein Sur7 and gold nanoparticles visible under an electron microscope, researchers showed that the protein sits on the outside of some—but not all—of the vesicles shed by the pathogen, and that the labeled particles are measurably larger than their unlabeled counterparts.

Extracellular vesicles, or EVs, are small membrane-bound compartments ranging from roughly 30 to 1,000 nanometers in diameter that ferry proteins, lipids, nucleic acids and metabolites between cells. In pathogenic fungi, these particles are known to support virulence, transporting effector proteins, cell wall-degrading enzymes and even RNA molecules that can alter gene expression in the host.

The research team, led by Donovan Garcia-Ceron and colleagues including Marilyn A. Anderson, approached the problem by mining previously published proteomics data on F. graminearum EVs. They cross-referenced a list of 22 candidate EV markers identified in the yeast Candida albicans against the fungal pathogen’s vesicle proteome, searching for orthologous proteins using BLASTp. Thirteen of the 22 candidates turned up in the F. graminearum EV proteome. Of those, three proteins appeared exclusively in the vesicles, six were enriched there relative to the cell lysate, and four showed no difference between the two sample types. The team then narrowed the field to proteins that were either exclusive to or enriched in EVs and possessed transmembrane domains suitable for antibody targeting, landing on four candidates: Fet3, 1,3-β-glucanosyltransferase, Yop1 and Sur7.

Membrane topology predictions using two independent tools, Protter 1.0 and TOPCONS 2.0, produced inconsistent results for Fet3 and Yop1, so those candidates were dropped. The predictions for 1,3-β-glucanosyltransferase and Sur7 agreed between the two programs, but Sur7 had the stronger case: it had already been flagged as a potential marker of C. albicans EVs, and its predicted topology resembles that of mammalian tetraspanins, the protein family that serves as the workhorse markers of mammalian EV research. Sur7 is a tetraspanin-like protein with four transmembrane domains and two extracellular loops, and it had previously been shown to influence EV morphology, cargo composition and fungal virulence in C. albicans, where deleting the gene altered the vesicle proteome, produced smaller vesicles and attenuated virulence in a Galleria mellonella infection model.

To generate a tool capable of recognizing the protein, the researchers used the epitope prediction program Bepipred 3.0 to scan the full FgSur7 amino acid sequence for regions likely to provoke an antibody response. They selected a 24-amino-acid peptide—DGARAAPVIGKAWDSNPRNAPSS—located in the protein’s large extracellular loop, where its antigenicity score exceeded a threshold of 0.2. Mapping that peptide onto an AlphaFold-predicted three-dimensional structure of the protein confirmed it sat on the exposed outer surface. A synthetic version of the peptide, linked to the carrier protein keyhole-limpet hemocyanin, was injected into two rabbits, and the resulting polyclonal antibody was purified to a concentration of 1 milligram per milliliter.

Western blotting confirmed the antibody did its job. When applied to EV-enriched samples from F. graminearum, the anti-FgSur7 antibody produced a strong band just below the 28-kilodalton marker, consistent with Sur7’s theoretical molecular mass of 27 kilodaltons. The same band was not detected in cell lysate samples, although earlier proteomics work by the same group had established that Sur7 is enriched in vesicles relative to the whole-cell fraction. When the researchers pretreated vesicle samples with proteinase K, an enzyme that digests exposed proteins, the Sur7 signal disappeared—evidence that the epitope sits on the vesicle exterior rather than tucked inside the membrane. The antibody showed no cross-reactivity with EVs from Saccharomyces cerevisiae or C. albicans, and control blots with pre-immunization serum revealed only a non-specific band near 50 kilodaltons that appeared in both pre- and post-immunization samples.

The decisive experiment came with immunogold electron microscopy. The team placed vesicle samples on carbon-coated copper grids, blocked them with bovine serum albumin, and incubated them with the anti-FgSur7 antibody followed by a secondary antibody conjugated to 5-nanometer colloidal gold particles. Under the electron microscope, gold nanoparticles clustered on the surfaces of some vesicles but were absent from others, confirming that Sur7 is displayed on the outer membrane of a subset of F. graminearum EVs. Negative controls—no primary antibody, primary antibody pre-blocked with its epitope peptide, bovine serum albumin alone, and pre-immunization rabbit serum—produced no significant non-specific gold labeling, and wide-field micrographs confirmed the gold was concentrated around vesicular structures rather than scattered across the grid background.

Quantitative analysis of the micrographs added an unexpected dimension. Measuring particle diameters in Fiji, the researchers found that Sur7-positive vesicles averaged 209 nanometers in diameter (n = 40), while Sur7-negative vesicles averaged just 138 nanometers (n = 16). A Wilcoxon pair-wise test confirmed the difference was statistically significant, with a p-value of 0.0039. Kernel density estimation visualized the distinct size distributions of the two populations. The size difference echoes what has been reported in mammalian systems, where medium-sized EVs carry higher proportions of the tetraspanins CD9 and CD81 while smaller EVs carry more CD63, correlating with where those proteins reside in the cell—CD9 and CD81 in the plasma membrane, CD63 in endosomes. Since Sur7 is predicted to be a plasma membrane protein, the authors speculate it may mark vesicles that bud directly from the cell surface, though they caution that its presence in vesicles of other origins cannot be ruled out.

Sequence analysis extended the finding beyond a single species. The Sur7 epitope used for antibody production is fully conserved—save for a single-base mutation—across multiple Fusarium species, and is at least 85 percent identical in 65 Fusarium species examined. Fungi from the genera Cylindrodendrum, Ilyonectria, Clonostachys, Neonectria and Stachybotrys share roughly 70 percent identity with the epitope. By contrast, the corresponding region in C. albicans and S. cerevisiae shows no significant sequence conservation, which explains the antibody’s lack of cross-reactivity with those organisms’ vesicles. A phylogenetic tree built from Sur7 orthologs across a dozen fungal pathogens—including Magnaporthe grisea, Zymoseptoria tritici, Botrytis cinerea and Fusarium oxysporum—confirmed that Fusarium sequences cluster together and apart from non-Fusarium fungi. The authors suggest Sur7 could therefore serve as a pan-Fusarium EV marker, and the antibody might even find use as a protein-based diagnostic complement to existing mycotoxin-based detection tools, particularly for Fusarium isolates that cause crop damage without producing toxins.

The practical implications reach further than taxonomy. With a validated surface marker in hand, researchers can now attempt immunoaffinity chromatography to physically separate Sur7-positive from Sur7-negative vesicle subpopulations, track vesicles as they move from the fungal cell through the cell wall and into host tissue using immunogold microscopy, and label fungal EVs alongside plant EVs marked with homologs of the Arabidopsis proteins PEN1 and TET8. The authors also propose generating SUR7-GFP transgenic lines to visualize potential sites of vesicle biogenesis inside the fungal cell, which could help confirm whether fungal EVs originate from multivesicular bodies, direct plasma membrane budding, or both. Beyond F. graminearum, the team notes that other proteins exclusive to the pathogen’s vesicles—Cdc42, Rho3, Ykt6-like and the structurally unresolved Yop1—remain promising antibody targets. If EV production can eventually be disrupted or redirected in crop pathogens, the vesicle surface itself might even become a target for crop protectants that trigger plant defenses in the absence of infection, a hypothesis the authors say can now be tested using the antibody to track how vesicles interact with host tissue.

Subject of Research: Surface protein markers on extracellular vesicles of the fungal wheat pathogen Fusarium graminearum

Article Title: Immunogold labeling of extracellular vesicles from the fungal pathogen Fusarium graminearum reveals the presence of the protein marker Sur7

Article References: Garcia-Ceron, D., Wilson, S. M., Bleackley, M. R., & Anderson, M. A. (2025). Immunogold labeling of extracellular vesicles from the fungal pathogen Fusarium graminearum reveals the presence of the protein marker Sur7. Fungal Biology and Biotechnology, 13(1), Article 2. https://doi.org/10.1186/s40694-025-00206-8

Image Credits: AI Generated

DOI: 10.1186/s40694-025-00206-8

Keywords: Fusarium graminearum, extracellular vesicles, Sur7, immunogold labeling, fungal pathogen, tetraspanin, EV markers, Western blot, electron microscopy, antifungal targets, wheat head blight, polyclonal antibody