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.
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)
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.
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.
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.