RXLR-Mediated Delivery of Exogenous CRr Protein Reduces Clubroot in Tumorous Stem Mustard and Reshapes the Rhizosphere Microbiome

Clubroot, incited by the obligate biotrophic protist Plasmodiophora brassicae, poses a severe threat to cruciferous crop production worldwide. Building on the concept of exogenous NLR protein delivery, we tested whether a bacterially expressed CRr protein (a recombinant Crr1a protein fused with an RXLR motif) could confer protection against clubroot in tumorous stem mustard (Brassica juncea var. tumida). Seedling roots were exposed to a crude lysate harboring the RXLR–Crr1a chimera, after which the fate of the protein and its downstream consequences were monitored across molecular, ecological, and metabolic levels. Time-resolved fluorescence imaging demonstrated that the recombinant signal became broadly distributed throughout the root system within a 24 h window, concomitant with a marked alleviation of disease severity. An empty vector control (equal protein, pET-28a(+) lysate) showed no significant protection versus the pathogen-only control (p > 0.05), suggesting that the observed protection is associated with the CRr protein lysate rather than general bacterial components. At the microbial level, the treatment fostered a more interconnected rhizosphere co-occurrence network and selectively promoted taxa, recognized as beneficial bacteria. At the metabolic level, several defense-associated compounds were elevated, among them jasmonic acid methyl ester, (2R)-2-hydroxyphenethylglucosinolate, 8-hydroxyluteolin 4′-methyl ether 8-glucuronide, and 4-hydroxyglucobrassicin. Furthermore, soil populations of beneficial bacteria including the Gram-positive genera Bacillus and Priestia exhibited strong positive associations with host defense metabolites. Collectively, these findings support a model in which externally supplied CRr protein lysate induces a response that can be observed at the metabolite and microbiome levels via rapid protein dissemination. This work lays a conceptual foundation for protein-based, microbiome-oriented management of soil-borne diseases without genetic modification of the crop.