The defense-inducing compound <i>N</i> -hydroxy pipecolic acid is a major substrate for the methyltransferase PbBSMT of <i>Plasmodiophora brassicae</i>

The clubroot pathogen Plasmodiophora brassicae has an effector protein that can methylate the plant defense compound salicylic acid (SA), and the protein sequence is highly conserved in different isolates worldwide. Sequencing the PbBSMT full-length cDNA from a small collection of clubs indicated the occurrence of a natural variation that differs by 18 amino acids, which was also found within the worldwide data collection for P. brassicae isolates. A search for more possible substrates was expanded to include defense-inducing compounds. Interestingly, PbBSMT converted N-hydroxy pipecolic acid (NHP), a recently reported inductor of systemic defense, also to its methyl ester. When the enzyme characteristics were compared, the protein showed much lower Km values for the substrates SA and NHP compared to the other two natural compounds previously identified to be methylated with high activities (benzoic and anthranilic acids). Therefore, NHP and SA seem to be the most favored substrates in vitro. There was no difference between the two variants in terms of being able to accept the four naturally occurring substrates. Incubating mature clubs 21 days after inoculation with NHP or SA also resulted in the in vivo conversion to their respective methyl esters. It was possible to detect the methyl ester of NHP specifically in transgenic plants overexpressing PbBSMT under inductive conditions. Arabidopsis thaliana mutants and transgenic lines with decreased or increased NHP levels were analyzed for their disease severity and the development of clubs. All plant lines with elevated NHP levels had reduced club weights, even though no reduction in infection rate or disease severity was seen in either of these lines. Treatments of infected plants with NHP did not reduce disease severity and therefore, a major function for NHP in defense against the clubroot pathogen is not apparent.