Expression and localization of nitrilase during symptom development of theclubroot disease in Arabidopsis

The expression of nitrilase in Arabidopsis during the development of the cl ubroot disease caused by the obligate biotroph Plasmodiophora brassicae was investigated. A time course study showed that only during the exponential growth phase of the clubs was nitrilase prominently enhanced in infected ro ots compared with controls. NIT1 and NIT2 are the nitrilase isoforms predom inantly expressed in clubroot tissue, as shown by investigating promoter-be ta-glucuronidase fusions of each. Two peaks of beta-glucuronidase activity were visible: an earlier peak (21 d post inoculation) consisting only of th e expression of NIT1, and a second peak at about 32 d post inoculation, whi ch predominantly consisted of NIT2 expression. Using a polyclonal antibody against nitrilase, it was shown that the protein was mainly found in infect ed cells containing sporulating plasmodia, whereas in cells of healthy root s and in uninfected cells of inoculated roots only a few immunosignals were detected. To determine which effect a missing nitrilase isoform might have on symptom development, the P. brassicae infection in a nitrilase mutant ( nit1-3) of Arabidopsis was investigated. As a comparison, transgenic plants overexpressing NIT2 under the control of the cauliflower mosaic virus 35S promoter were studied. Root galls were smaller in nit1-3 plants compared wi th the wild type. The phenotype of smaller clubs in the mutant was correlat ed with a lower free indole-3-acetic acid content in the clubs compared wit h the wild type. Overexpression of nitrilase did not result in larger clubs compared with the wild type. The putative role of nitrilase and auxins dur ing symptom development is discussed.