Isoform-Specific Localization of Brassica rapa Nitrilases in Root Infected with Plasmodiophora brassicae Revealed Using In Situ Hybridization Probes Improved with Locked Nucleic Acids

Abstract

We established an in situ hybridization (ISH) technique by modification of hybridization probes with locked nucleic acids (LNAs) and demonstrated isoform-specific localization of transcripts of Brassica rapanitrilase (BrNIT-T) genes in clubroot tissue infected with Plasmodiophora brassicae. Chimeric oligo DNA probes containing LNAs demonstrated highly improved specificities and could discriminate between BrNIT-T1 and BrNIT-T2. These LNA-containing probes were applied to ISH. BrNIT-T1 was strongly expressed in cells containing expanding secondary plasmodia of P. brassicae, but not in cells containing resting spores. On the other hand, BrNIT-T2 transcripts were localized in noninfected cells rather than infected cells during the clubroot growth phase but coexisted with mature resting spores at a later phase of clubroot development. Immunostaining for indole-3-acetic acid (IAA) revealed IAA accumulation in cells containing growing plasmodia. IAA immunostaining in infected cells was reduced as the pathogen formed resting spores, but the signal was again enhanced in cells containing mature resting spores at a later phase of infection, suggesting that IAA is involved in both the early growth and the latest maturation phase of clubroot development. Expression of BrNIT-T1 and BrNIT-T2 in turnip roots was upregulated by exogenous treatment with cytokinin and jasmonic acid, respectively. Thus, these two phytohormones are possible triggers of abnormal IAA production in clubroot tissue via induction of the respective nitrilase. Given these results, we propose a model for isoform-specific roles of B. rapa nitrilases in auxin biosynthesis involved in phytohormone crosstalk during development of clubroot disease.