Expression and Localization of Nitrilase during Symptom Development of the Clubroot Disease in Arabidopsis

Abstract

The expression of nitrilase in Arabidopsis during the development of the clubroot 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 roots compared with controls. NIT1 and NIT2 are the nitrilase isoforms predominantly expressed in clubroot tissue, as shown by investigating promoter-␤-glucuronidase fusions of each. Two peaks of ␤-glucuronidase activity were visible: an earlier peak (21 d post inoculation) consisting only of the expression of NIT1, and a second peak at about 32 d post inoculation, which predominantly consisted of NIT2 expression. Using a polyclonal antibody against nitrilase, it was shown that the protein was mainly found in infected cells containing sporulating plasmodia, whereas in cells of healthy roots 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 with the wild type. The phenotype of smaller clubs in the mutant was correlated with a lower free indole-3-acetic acid content in the clubs compared with the wild type. Overexpression of nitrilase did not result in larger clubs compared with the wild type. The putative role of nitrilase and auxins during symptom development is discussed.

Genetic Analysis of Clubroot Resistance in Brassica Crops

“…Increase in indole-3-acetonitrile nitrilase activity, which cleavages indole-3-acetonitrile to indole-3-acetic acid is demonstrated by several authors (Raush et al 1981, Ugajin et al 2003. The expression of nitrilase genes during club formation was also demonstrated (Grsic- Rausch et al 2000). Microarray techniques are now used to understand the mechanism of club formation (Ludwig-Müller 2004).…”

Auxin homeostasis, signaling, and interaction with other growth hormones during the clubroot disease of Brassicaceae

“…In line of these findings, the nitrilase protein of Arabidopsis, involved in IAA biosynthesis, has been localized also in cells harboring large plasmodia by immunohistochemical methods. 9 It is interesting to note that GH3.5 shows high expression levels at the second time point (3 weeks after inoculation). Since the protein GH3.5 is also able to conjugate salicylic acid to amino acids 10 and since it is involved in the biosynthesis of camalexin, 11 its specific regulation warranted further analysis.…”

Differential Regulation of Root Arginine Catabolism and Polyamine Metabolism in Clubroot-Susceptible and Partially Resistant Arabidopsis Genotypes

“…Consequently, knowledge acquired on Arabidopsis could then be rapidly integrated and transferred to cultivated crops. To date, research on clubroot using Arabidopsis as a model host system was mainly focused on the role of hormonal regulation by auxin (Ludwig-Muller et al, 1999;Grsic-Rausch et al, 2000;Neuhaus et al, 2000;Ando et al, 2006;Devos et al, 2006;Schuller and LudwigMuller, 2006) and/or cytokinins (Devos et al, 2006;Siemens et al, 2006) in the development of clubroot symptoms. However, other molecules, such as polyamines, could also be involved in cell proliferation and differentiation.…”