Plasmodiophora brassicae, the causal agent of clubroot disease, actively manipulates host hormone pathways to promote infection. Previous studies identified PbGH3 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 PbGH3 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 PbGH3 in the gh3.12 mutant background partially restored SA accumulation and reduced disease susceptibility. PbGH3 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.
Clubroot disease, caused by the obligate biotrophic pathogen Plasmodiophora brassicae, is a major threat to canola (Brassica napus) production worldwide. Clubroot-resistant (CR) cultivars remain the most effective disease-management strategy, but the genetic basis of resistance in commercial canola remains poorly understood because many resistance sources are proprietary and associated genotypic information is rarely accessible. Although nucleotide-binding leucine-rich repeat (NLR) immune receptors account for most cloned CR genes, no pan-NLRome has incorporated CR lines used in commercial canola breeding. Here, we combined whole-genome sequencing and resistance gene enrichment sequencing (RenSeq) to assemble and annotate the NLR repertoires of five homozygous CR inbred lines (IH1–IH5) used for commercial breeding and displaying contrasting resistance profiles against predominant Canadian P. brassicae pathotypes. We integrated these NLRomes with the susceptible cultivar Westar to construct a comparative pan-NLRome for canola. Across the five CR lines, total NLR content was highly conserved, ranging from 504 to 517 genes, with TIR-NLRs representing the predominant class. C-JID-containing TIR-NLRs accounted for more than 30% of each NLR repertoire, and integrated-domain analysis identified conserved and genotype-specific NLR-IDs, including previously unreported domains in IH4. Pan-NLRome analysis resolved 366 NLR orthogroups (OGs), 60.7% of which were core, and identified resistant-line-enriched OGs absent from Westar as candidate CR-associated loci. Unexpectedly, a homolog of the functionally characterized CR gene, CRa, was detected in five CR lines. Moreover, a homolog of another CR gene, Crr1a, was detected in both resistant and susceptible lines, indicating that the presence/absence of a gene alone does not predict resistance. Instead, structural variation affecting LRR and C-JID regions suggests that allele-level diversity within conserved NLR loci contributes to CR-associated variation, with implications for allele-specific marker development and durable CR deployment.
Jiaxu Wu, Soham Mukhopadhyay, Muhammad Asim Javed et al.· bioRxiv· 0 citations
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