A Clubroot Pathogen PBS3-Like Effector Manipulates Hormonal Crosstalk to Alter Root Morphology in Arabidopsis and Canola

Plasmodiophora brassicae, the causal agent of clubroot disease, actively manipulates host hormone pathways to promote infection. Previous studies identified PbGH ₃ as a putative GH3-like effector with in vitro auxin-conjugating activity, although its biological function in planta was investigated but remained unresolved. Here, we studied the role of PbGH3 during host colonization using PbGH ₃ overexpressing Arabidopsis thaliana and Brassica napus lines. PbGH3 overexpression induced conserved developmental phenotypes in both species, including epinastic leaves, reduced apical dominance, enhanced lateral branching, and increased root hair formation, consistent with altered hormone balance. However, hormone profiling and exogenous auxin assays did not support a role for PbGH3 as a canonical auxin-conjugating enzyme in planta. Instead, structural analyses revealed strong similarity between PbGH3 and the Arabidopsis clade III GH3 proteins GH3.12/PBS3 and GH3.7, which are associated with salicylic acid (SA) metabolism. Consistent with this observation, loss of GH3.12/PBS3 increased susceptibility to clubroot, while expression of PbGH ₃ in the gh3.12 mutant background partially restored SA accumulation and reduced disease susceptibility. PbGH ₃ expression peaked during the early stages of infection, and overexpressing lines displayed enhanced root hair colonization by P. brassicae, suggesting a role during primary infection rather than later gall development. Together, our results support a model in which PbGH3 modulates SA-associated hormonal crosstalk and root morphology that could be harnessed by the clubroot pathogen to ensure host colonization. These findings provide new insight into how P. brassicae manipulate host hormone homeostasis during infection.