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Plasmodiophora brassicae host-parasite-interaction (Johannes Siemens)

The group is examining the interaction of the obligate root parasite Plasmodiophora brassicae Wor. with its host plants in the genus Brassica and the model plant A. thaliana (L.) Heynh. Thereby is the resistance gene RPB1 of A. thaliana on side of the host and the corresponding virulence genes on side of the parasite in focus of analysis.

Description of the disease development

Cloning of the resistance gene RPB1

Virulence genes of the Protist P. brassciae

Bachelor, Diploma und Ph.D. theses  


Additional topic in History of science: Lyssenkoism in Germany  

Additional Topic in Politics of Science: Green Gene technology  

1. Description of the pathogenesis

Based on an extended histological description we have worked out a good knowledge of the development of the disease as well as the resistance reaction in ecotypes of A. thaliana (Kobelt et al. 2000). The extension of these studies to immunohistochemical analysis resulted in the analysis of the role of the enzym nitrilase during infection (Grsic et al. 1998, 2000) and the examination of the cytoskeleton of P. brassicae using confocal laser scanning microscopy (Kobelt et al. in Prep.). This detailed description of the pathogenesis was the basis of different measurements to quantify disease symptoms for comparison of different  A. thaliana mutant lines, which might influence the development of the disease (Siemens et al. 2002).

An initial molecular analysis was done by small genomic libraries of P. brassicae (Klewer et al. 2001) and continued by subtractive cDNA libraries of susceptible and resistant ecotypes infected or not infected with P. brassicae (Arbeiter et al. 2002). To obtain more insights into clubroot disease these works were extended by a microarray-experiment using the Arabidopsis full genome chip (Affymetrix) in cooperation with Martin Parniske at the John Innes Centre (Norwich, GB) and Jutta Ludwig-Müller (Dresden, FRG). The analysis of the microarray-data (ArrayExpress Accessions Nr. E-MEXP-254) is still ongoing. Highly up- or down-regulated genes will be studied by RT-PCR to evaluate the microarray data and by transgenic lines to obtain initial hints of the importance of regulated genes for the disease (in part topic of the Ph.D. thesis of Cornelia Horn). Such work has already been done for the cytokinin-oxidase/dehydrogenase (CKX) genes in cooperation with the group of Thomas Schmülling (Berlin, FRG). Transgenic lines overexpressing CKX I or CKX III reveal a significantly reduced susceptibility, thereby indicating the importance of the down-regulation of CKX host genes during the initial phase of clubroot disease combined with cytokinin production by the pathogen (Siemens et al., submitted).

In similar experiments the roles of different genes are currently studied during clubroot infection, e.g. the cytokinin-receptors in cooperation with Michael Riefler (Berlin, FRG), the invertases and invertase-inhibitores in cooperation with Thomas Roitsch (Würzburg, FRG), Thomas Rausch (Heidelberg, FRG) and Jutta Ludwig-Müller (Dresden, FRG), and phytoalexins and glucosinolates in cooperation with Erich Glawischnig (München, FRG).

Schematic view  of our current hypothesis of the clubroot disease development (annotated).

The extension of microarray-analysis to further experiments is discussed with the groups of Maria Manzanares-Dauleux (AgroCampus Rennes, France) and Robert Faggian (Melbourne, Australia). The data basis will be strongly improved by combination of all these experiments and the chance to find crucial pathogenesis-relevant genes will be increased.

grant (since july 2004)
Sächsisches Landesamt für Umwelt und Geologie (LfUG)
Production of root-gall resistance by pathogenesis-inverse direction of the hormone metabolim.
in cooperation with Jutta Ludwig-Müller (Dresden, FRG)

2.  Cloning of the resistance gene RPB1 of A. thaliana

Allels of the resistance gene RPB1 have been found in four ecotypes of A. thaliana (Kobelt et al. 2000, Arbeiter et al. 2002). The gene has been localised by mapping in a region of  70 kb on chromosome 1 (Arbeiter et al. 2003). According to the genome data of the susceptible ecotype
Columbia in this region 14 candidate gene are localised, which are mainly annotated as genes of unknown function with low homology to known proteins. None of these candidate genes reveals characteristics of known resistance genes (Arbeiter et al. 2003). At the moment these candidate genes were transformed to susceptible ecotypes to obtain biological evidence for the function of these genes. As part of his Ph.D. thesis Frank Rehm is working on transformation of these genes.

Meanwhile the gene has been studied by crossing with mutant lines of salicylic acid, jasmonic acid and ethylene metabolism to characterise the signal transduction chain of RPB1 (Arbeiter et al. 2002). The segregation data of these crossings indicate no importance of these phytohormones in the resistance response, indicating an integration of  RPB1 into an unusual signal transduction chain.  In cooperation with Jane Parker (Köln, FRG) we currently analyse the role of the genes sgt1a, sgt1b and rar in the resistance response to Plasmodiophora.

grant (since june 2004)
Deutsche Forschungsgemeinschaft (Si706/5-1)
Characterisation of the resistance gene RPB1 of Arabidopsis thaliana to Plasmodiophora brassicae

3. Virulence genes of the pathogen P. brassicae

On side of the pathogen we take advantage of the interaction of A. thaliana and P. brassicae to characterize single-spore isolates under controlled conditions and defined genetic background of host and pathogen (Klewer et al. 2001, Siemens et al. 2003). Virulence genes of P. brassicae can be distinguished using the interaction with host plants. The group has established molecular fingerprints using repetitive elements of P. brassicae (Klewer et al. 2001, Fähling et al. 2003) and electrophoretic karyograms (Graf et al. 2001, Graf et al. 2004) to further characterisation of isolates.

Using these tools the selection of virulent  pathotypes in resistant Brassica-plants (Fähling et al. 2003) and the reorganisation of chromosomes of the pathogen after propagation in host plants (Fähling et al. 2004) has been proven. Different isolates of P. brassicae show chromosome ­polymorphism (Graf et al. 2004), although these polymorphism cannot be used to estimate a virulence pattern. However, the group has established a set of related isolates, which might provide insights into virulence pattern.
These isolate shall be characterised by two-dimensional pulsed field electrophoresis and further molecular fingerprints using gene fragments isolated from the genomic or subtractive cDNA-libraries  (see 1).

Beside differentiation of isolates the group analyses the expression pattern of genes of the pathogen in cooperation with Jutta Ludwig-Müller (Dresden, FRG) and Simon Buhlman (Christchurch, New Zealand). Most isolated genes of Plasmodiophora show no or only poor homology to known genes. Up to date those genes can only be studied by expression pattern and sequence analysis. However, expression patterns of host and pathogen genes during disease development might provide landmarks to describe the disease in more detail.

Last modified: 06.05.2008 16:49
Author: Johannes Siemens

Johannes Siemens
Tel.: +49 351 463-36789
Fax: +49 351 463-37749
Mail an Johannes.Siemens@tu-dresden.deJohannes.Siemens@tu-dresden.de

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Technische Universität Dresden
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Molecular Biotechnology
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