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Updated: Aug 13, 2026
Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag
Published on: November 1, 2018
Affinity-tags are removed from Cladosporium fulvum effector proteins expressed in the tomato leaf apoplast
H Peter van Esse1, Bart P H J Thomma, John W van 't Klooster
1Laboratory of Phytopathology, Centre for Biosystems Genomics (CBSG), Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands.
Abstract:
Cladosporium fulvum (syn. Passalora fulva) is a biotrophic fungal pathogen that causes leaf mould on tomato (Solanum esculentum). The fungus grows exclusively in the tomato leaf apoplast where it secretes several small (<15 kDa) cysteine-rich proteins that are thought to play a role in disease establishment. To investigate the role of these proteins, and to identify their in planta targets, a targeted proteomics approach was undertaken. C. fulvum proteins were expressed as recombinant fusion proteins carrying various affinity-tags at either their C- or N-terminus. Although these fusion proteins were correctly expressed and secreted into the leaf apoplast, detection of affinity-tagged C. fulvum proteins failed, and affinity purification did not result in the recovery of these proteins. However, when using C. fulvum effector protein-specific antibodies, specific signals were obtained for the different proteins. It is concluded that the stability of the in planta expressed recombinant fusion proteins is insufficient, which results in removal of the affinity-tag from the fusion proteins, irrespective of the C- or N-terminal fusion or the nature of the affinity-tag. Similar phenomena were observed when the fusion proteins were expressed in other Solanaceous species, but not when expressed in Arabidopsis thaliana.
Insights
Recombinant Cladosporium fulvum proteins expressed in tomato leaves were unstable, leading to affinity-tag removal. This instability hindered the identification of fungal effector targets in planta.
Area of Science:
- Plant Pathology
- Molecular Biology
- Mycology
Background:
- Cladosporium fulvum causes tomato leaf mould by secreting small effector proteins into the apoplast.
- Understanding these effectors is crucial for identifying their roles in disease establishment and potential targets within the host plant.
Purpose of the Study:
- To investigate the function of C. fulvum effector proteins.
- To identify in planta targets of these secreted fungal proteins using a targeted proteomics approach.
Main Methods:
- Expression of C. fulvum proteins as N- or C-terminal affinity-tagged fusion proteins in planta.
- Attempted detection and purification of tagged proteins using affinity chromatography.
- Utilized specific antibodies against C. fulvum effector proteins for detection.
Main Results:
- Fusion proteins were expressed and secreted into the tomato leaf apoplast.
- Detection and affinity purification of tagged proteins failed due to apparent tag removal.
- Specific signals were detected using effector protein-specific antibodies, indicating protein presence.
Conclusions:
- In planta expressed recombinant fusion proteins exhibit insufficient stability, leading to affinity-tag detachment.
- This instability complicates the identification of effector targets in susceptible Solanaceous species.
- Expression in Arabidopsis thaliana did not show similar tag removal, suggesting species-specific effects.
Related Concept Videos
01:24Tagging and Fusion Proteins
01:03Affinity Chromatography