TurboID-based proximity labeling enables in vivo mapping of Plasmodiophora brassicae secretome in Arabidopsis

O_LIPlasmodiophora brassicae, the causal agent of clubroot disease, is an obligate biotrophic protist belonging to the poorly characterized Rhizaria. Its intracellular lifestyle and resistance to genetic manipulation have hindered functional analysis of its effector repertoire, leaving mechanisms underlying disease development unresolved. Here, we sought to experimentally define the P. brassicae secretome within infected plant cells and identify effectors targeted to specific host subcellular compartments. C_LIO_LIA proximity labeling approach based on the TurboID biotin ligase was used to capture pathogen-derived proteins within the nucleus, cytosol, endoplasmic reticulum, and plasma membrane of infected Arabidopsis roots during primary and secondary stages of clubroot disease. C_LIO_LIThis strategy yielded the first in planta experimental view of the P. brassicae secretome, identifying both established and previously uncharacterized effectors. The resulting dataset provides a valuable resource and methodological framework for dissecting effector function in this and other intracellular plant pathogens. C_LIO_LIThis study expands our understanding of Rhizarian pathogenicity and provides a methodological template for identifying the secretomes of other obligatory intracellular plant pathogens. C_LI