The clubroot disease of the family Brassicaceae is caused by the obligate biotrophic protist Plasmodiophora brassicae. Infected roots undergo a developmental switch that results in the formation of aberrant roots (clubs). To investigate host gene expression during the development of the disease, we have used the Arabidopsis ATH1 genome array. Two timepoints were chosen, an early timepoint at which the pathogen has colonized the root but has induced only very limited change of host cell and root morphology and a later timepoint at which more than 60% of the host root cells were colonized and root morphology was drastically altered. At both timepoints, more than 1,000 genes were differentially expressed in infected versus control roots. These included genes associated with growth and cell cycle, sugar phosphate metabolism, and defense. The involvement of plant hormones in club development was further supported; genes involved in auxin homeostasis, such as nitrilases and members of the GH3 family, were upregulated, whereas genes involved in cytokinin homeostasis (cytokinin synthases and cytokinin oxidases/dehydrogenases) were already strongly downregulated at the early timepoint. Cytokinin oxidase/dehydrogenase overexpressing lines were disease resistant, clearly indicating the importance of cytokinin as a key factor in clubroot disease development.
The soil‐borne obligate pathogen Plasmodiophora brassicae causes clubroot disease in species of Brassicaceae, including Arabidopsis thaliana. The host–pathogen interaction was studied with respect to the age of the plant at the time point of inoculation and to different infection pressures in order to establish a standardization of infection parameters and evaluation of disease extent for A. thaliana lines. Spore number per root weight, root and shoot weight of inoculated and non‐inoculated plants as well as infection rate and disease index (DI) were analysed and correlated. The disease extent of different lines was comparable as measured by the relation of root weight of inoculated and non‐inoculated plants (Ri/Rni index) and the DI. Most of the 71 screened A. thaliana lines turned out to be susceptible. However, the mutant lines tu8, tu3, det1‐1, and rhd3‐1 showed a certain degree of tolerance under specific culture conditions. The reactions of rhd3‐1 indicate that hypertrophy is a prerequisite for maturation of the pathogen. The reactions of the tu3 and tu8 mutants indicate a role of indole glucosinolates and indole‐3‐acetonitrile/IAA in development of clubroot disease.
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.
Clubroot disease of Brassicaceae is caused by an obligate biotrophic protist, Plasmodiophora brassicae. During root gall development, a strong sink for assimilates is developed. Among other genes involved in sucrose and starch synthesis and degradation, the increased expression of invertases has been observed in a microarray experiment, and invertase and invertase inhibitor expression was confirmed using promoter::GUS lines of Arabidopsis thaliana. A functional approach demonstrates that invertases are important for gall development. Different transgenic lines expressing an invertase inhibitor under the control of two root-specific promoters, Pyk10 and CrypticT80, which results in the reduction of invertase activity, showed clearly reduced clubroot symptoms in root tissue with highest promoter expression, whereas hypocotyl galls developed normally. These results present the first evidence that invertases are important factors during gall development, most probably in supplying sugars to the pathogen. In addition, root-specific repression of invertase activity could be used as a tool to reduce clubroot symptoms.
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