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

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 full-length cDNA of P. brassicae Benzoic acid/Salicylic acid Methyltransferase (PbBSMT) 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 including defense-inducing compounds revealed that PbBSMT was also able to convert a recently reported inductor of systemic defense, N-hydroxy pipecolic acid (NHP), to its methyl ester. PbBSMT showed much lower Km values for the substrates SA and NHP compared with the other two natural compounds previously identified to be methylated with high activities (benzoic and anthranilic acids), suggesting they are the most favored substrates in vitro. The two variants showed no difference in terms of being able to accept the four naturally occurring substrates. Mature clubs incubated for 21 d after inoculation with NHP or SA showed in vivo conversion to their respective methyl esters. The methyl ester of NHP was specifically detected in transgenic plants overexpressing PbBSMT under inductive conditions. Transgenic Arabidopsis with elevated NHP levels had reduced club weights, but no reduction in infection rate or disease severity was seen in these plants, or in mutants with decreased NHP levels. Treatment 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.