The GH3 Protein from the Clubroot Pathogen Plasmodiophora brassicae Causes Hormone-Related Phenotypes in Arabidopsis thaliana

Abstract

In the genome of the obligate biotrophic protist Plasmodiophora brassicae, which causes clubroot disease in the Brassicaceae family, a single gene was identified with homology to the group of GH3 proteins synthesizing amino acid conjugates with carboxylic acids. In comparison to plant members the P. brassicae GH3 protein seems to be very promiscuous with respect to the substrates converted. In vitro assays showed conversion of three different auxins (indole-3-acetic, IAA; indole-propionic, IPA; and indole-3-butyric acids, IBA), jasmonic acid (JA) and also 12-oxo-phytodienoic acid (OPDA). Auxin and Methyl-JA responsive elements were found in the promoter sequence of PbGH3. For further analysis Arabidopsis thaliana was transformed with PbGH3. Seedlings with confirmed constitutive PbGH3 protein production were grown to adult plants that were shown to produce fewer inflorescences per plant. Growth on 10 µM IAA or JA levels also altered the phenotype of the seedlings, but not so much between wild types and transgenic lines. The hormone profiles in seedlings and adult plants were examined for IAA, JA, OPDA, and respective conjugates. Transgenic seedlings displayed changes that could be partially attributed to the overexpression of a GH3 gene like lower levels of free IAA and the JA precursor cis-OPDA under control conditions, and higher levels of some auxin conjugates accumulated after IAA treatment compared to the corresponding wild types. However, the hormone patterns in adult plants were more complex and varied based on different tissue types analyzed. After inoculation with P. brassicae the transgenic plants did not show any changes in infection rate and disease index, but had higher shoot and lower root weight in infected plants compared to controls. Based on the above findings we discuss a role for PbGH3 during pathogenesis.