Interactions that take place in the plant apoplast are central to recognition of pathogen-derived molecules and activation of immune responses. The apoplast is an extracellular, often harsh environment that complicates biochemical analysis of protein–protein interactions. Proximity labelling approaches based on engineered biotin ligases, notably TurboID, have been widely adopted for mapping protein proximity and interactions inside cells. Prior to this report, TurboID applications in plants had been restricted to intracellular compartments. The authors aimed to adapt and validate a TurboID-based proximity labelling (PL) method for direct use in the leaf apoplast to enable detection of extracellular protein interactions.
To permit extracellular PL, the study deployed secreted versions of TurboID fused to apoplast-targeted proteins. The approach required (1) engineering TurboID fusions that are secreted to the apoplast, (2) confirming that fusion does not perturb biological activity or recognition by cognate receptors, and (3) providing biochemical conditions that allow TurboID enzymatic activity extracellularly. The well-characterised interaction between the oomycete elicitor INF1 from Phytophthora infestans and the receptor-like protein REL in Nicotiana benthamiana was selected as a test case to evaluate functionality in a biologically relevant apoplastic interaction.
Two secreted TurboID fusion constructs were used. SP-INF1-TurboID (abbreviated INF1-T) contains the INF1 elicitor sequence fused to TurboID and a signal peptide for secretion. A control construct, SP-eGFP-TurboID (eGFP-T), comprises secreted eGFP fused to TurboID. These constructs were transiently expressed in N. benthamiana leaves to test both localisation and labelling capability in the apoplast.
Functionality of the INF1 fusion was assessed by its ability to elicit the expected biological response. Transient expression of INF1-T induced a cell death (CD) response comparable to that triggered by native INF1, indicating that attaching the TurboID tag did not impair recognition of INF1 by REL. This preserved activity supports the use of the fusion as a physiologically relevant bait for proximity labelling of extracellular interactors.
The study confirmed apoplastic localisation of both INF1-T and eGFP-T, validating that the constructs reach the extracellular compartment targeted for interaction mapping. Apoplastic localisation was therefore suitable for carrying out TurboID-mediated labelling experiments in the leaf extracellular space.
Successful TurboID activity requires supply of substrate and cofactors. In the extracellular context, the authors achieved efficient TurboID-mediated biotinylation in the apoplast by co-infiltrating exogenous biotin along with ATP and magnesium acetate. These additions supported enzyme activity in the apoplast where endogenous availability of substrates and cofactors may be limited.
Proteins biotinylated by TurboID in apoplastic extracts were detected by streptavidin-HRP immunoblotting. Distinct biotinylation profiles were observed for INF1-T and eGFP-T, consistent with the expectation that different secreted bait proteins label different proximal protein environments in the apoplast.
To test whether the method could detect a known extracellular receptor, co-immunoprecipitation experiments were performed. These demonstrated specific biotinylation of REL by INF1-T but not by the control eGFP-T. This specific labelling occurred in wild-type N. benthamiana and in bak1 and sobir1/sobir1-like genetic backgrounds, indicating that INF1-T can selectively biotinylate its receptor in diverse host genotypes under the conditions tested.
The findings establish that TurboID-based proximity labelling can function in the plant apoplast and can be used to detect an elicitor–receptor interaction extracellularly. The preserved biological activity of INF1 when fused to TurboID, apoplastic localisation of the constructs, the requirement for exogenous biotin and cofactors, and specific biotinylation of REL together provide a proof-of-concept for deploying TurboID to interrogate extracellular interactions in leaves.
This report is presented as a preprint and has not been peer reviewed. The abstract and associated summary describe the experimental design, key observations and the proof-of-concept outcome; additional experimental details, quantitative metrics and broader validation experiments were not reported in the provided source summary. The authors declared no competing interests.