Exogenous Application of an RXLR-Fused CRa Protein Systemically Colonizes Roots and Suppresses Clubroot via Rhizosphere Bacterial Community Remodeling in Tumorous Stem Mustard

Clubroot, caused by the obligate biotrophic protist Plasmodiophora brassicae, is a devastating soil-borne disease threatening global cruciferous crop production. To explore non-transgenic control strategies, this study investigated the efficacy of a crude protein extract containing RXLR motif-fused recombinant CRa protein against clubroot in tumorous stem mustard (Brassica juncea var. tumida) and its regulatory mechanisms in the rhizosphere. Mechanistically, fluorescence tracking confirmed that the CRa protein exploits the xylem stream for systemic translocation, colonizing the stele and vascular tissues within 24 h and significantly reducing the disease index. Ecologically, CRa protein reshaped the rhizosphere microbiome by enhancing α-diversity (Shannon and Simpson indices), increasing network complexity, and specifically enriching beneficial genera such as Pseudomonas. Metabolically, CRa protein induced the reprogramming of root exudates, upregulating defensive compounds including jasmonic acid methyl ester, 4-hydroxyglucobrassicin, and (2R)-2-hydroxy-2-phenethylglucosinolate. Overall, exogenous CRa protein activates a coordinated defense response through three key mechanisms: rapid vascular translocation, recruitment of beneficial microbiota, and metabolic reprogramming. This study provides novel insights for developing non-transgenic, microbiome-based green control strategies.