Group leader: Dr.
rer. nat. Carin Jansen
Coworkers: Rebekka
Fensch,
Dipl. biol. Sibylle von
Rüden (PhD student), Dr. agr. Cléberson
Fernandes,
Lenka Malinowski
(Master student)
The Project GABI-Agrotec, which is funded by the
BMBF, was initiated in April 2002 as part of the national program GABI
(Genome analysis in the biological system plant). Besides the IPAZ (Institute
of Phytopathology and Applied Zoology, JLU Giessen), the Department of
Molecular Phytopathology and Genetics (Prof. Willi Schäfer, University
of Hamburg) and the Institute of Plant Genetics and Crop Plant Research
(Prof. Uwe Sonnewald, IPK Gatersleben) are working in the cooperative project
GABI-Agrotec. Aim of the project is the isolation and functional analysis
of barley genes, which contribute to the resistance of cereals to fungi
of the genus Fusarium.
Fungi of the genus Fusarium are so called
field fungi and lead to considerable yield losses in middle Europe, especially
in wheat but also in corn cultivation. The typical symptom of a Fusarium
infection in the wheat field is the partial or total head blight (Figure
1). But the reason why fungi of the genus Fusarium are in the focus
of scientific interest lies not only in the reduction of crop yield, but
also in the production of toxins. These mycotoxins can also be found in
foods and feeding stuff and can lead to severe health problems.
Because none of the presently available fungicides
provides a satisfying protection against Fusarium fungi, plant breeders
and phytopathologists both strive to solve the Fusarium problem.
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Figure 1. Partial and total head blight, respectively,
on wheat caused by a Fusarium infection. The growth of the fungus
inside the rachis leads to a damage of the tissue in such a manner, that
the nutrient transport into upper parts of the ear is aborted This part
of the ear bleaches and consequently dies.
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Isolation of candidate genes via cDNA-arrays
The contact between a plant and a pathogen always
leads to the activation of defence mechanisms, which should prevent the
spreading of the pathogen and development of disease symptoms. The speediness
and strength of distinct reactions often decide about the success of plant
defence. One precondition for all defence processes is the activation of
resistance associated genes, which products may for instance exhibit anti
fungal effects or prevent the access of the pathogen into the plant tissue
For that reason, GABI-Agrotec aims at the identification
of genes of the model plant barley (Hordeum vulgare L.),
which are activated during infection with Fusarium fungi in spikes,
roots and leaves, i.e. which expression is enhanced in comparison to the
uninfected status. These genes are found via so called cDNA-arrays (Figure
2). For this purpose, nearly 5,000 gene fragments were spotted onto a nylon
membrane, which is hybridized with probes from Fusarium infected
and non infected plant material.
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Figure 2. Cutout of a cDNA-array, which contains
nearly 5,000 cDNA fragments on an area of approx. 9 x 13 cm The activity
of the corresponding genes in infected and uninfected tissue, is determined
by computational analysis.
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The comparison of the expression profiles of the genes in
Fusarium
infected an uninfected barley spikes, leaves and roots leads to the identification
of genes, which expression is enhanced during the Fusarium infection.
Gene function assays to identify determinants of
resistance: The Arabidopsis-Fusarium pathosystem
Because stable transformation of cereals is longsome
and requires a lot of space, we established a pathosystem for a quicker
functional analysis of the candidate genes, using Arabidopsis thaliana
as host for Fusarium. Due to the high homology between barley and
Arabidopsis, for most of the barley genes Arabidopsis homologues can be
found. For many of the Arabidopsis genes, insertion lines exist, i.e. mutants
in which the candidate gene is no longer functional. This insertion lines
can easily be ordered at public stock centres. Besides F. oxysporum
strains known from literature, we also use the cereal pathogens F. graminearum
and F. culmorum in infection assays, because a successful infection
of Arabidopsis leaves by F. culmorum and of Arabidopsis roots by
F.
culmorum and F. graminearum could be established (Figure 3).
Figure 3. Generation of conidia by F. culmorum
on an Arabidopsis leaf, 8 days after artificial infection (A). Strong growth
of an A. th. root by a GFP-expressing strain of F. graminearum,
3 days after artificial infection (B).
Gene function assays to identify determinants
of resistance: Test of stably transformed cereals
Concluding, the involvement of the candidate genes
in the resistance to Fusarium will also be determined in functional
assays with cereals. For this purpose, the candidate genes are cloned into
special expression vectors and are transformed in wheat and barley immature
embryos using Agrobacterium tumefaciens. The different organs (spikes,
leaves, roots) of the transformed plants are inoculated with Fusarium
fungi and the comparison with the infection severity on untransformed plants
leads to the identification of those candidate genes, which have an impact
on the resistance to Fusarium.
The infestation of barley and wheat roots (Figure
4A) and leaves (Figure 4B) by Fusarium can be monitored quickly
and easily. The determination of the infection severity is carried out
macroscopically by measuring the necrotized area of the infected plant
organ. If the infestation of the transformed plant is reduced in comparison
to the untransformed control, detailed microscopic studies are conducted
to reveal possible changes on cytological level.
Figure 4 A/B. Infection of F. culmorum on
roots (A) and leaves (B) of barley. The infestation on the roots leads
to a brownish discoloration (necrotization) and a clearly visible reduction
of root length in comparison to the uninfected control, which is displayed
left hand side. On the leaves, Fusarium causes yellow-brownish lesions,
i.e. areas in which the plant tissue has died. Both pictures were taken
7 days after artificial infection.
The susceptibility of the transformed plants is also tested in spike
infection studies. Single wheat and barley spikes are infected with F.
graminearum by spraying a spore suspension on the spikes at anthesis.
After 3 weeks the percentage of infected spikelets is determined.
Study of selcted candidate
genes
Fungi of the genus Fusarium are necrotroph
pathogens, i.e. they actively kill the invaded plant tissue and feed on
the contents of dead cells. Due to this life style, it’s a thinkable strategy
to inhibit the fungus’ development by keeping the attacked or penetrated
plant cells alive. Several genes that have the capability to inhibit cell
death have been isolated from barely by date. These cell death inhibitors
should also be tested for a resistance-mediative effect in the cereal-Fusarium
pathosystem.
Cytological study of the
cereal-Fusarium interaction
Only
little is known about cytological details of the cereal-Fusarium
interaction. This may be due to the fact that microscopic analysis of the
fungus is quite complicated, because of the excessive generation of extracellular
mycelium and intracellular hyphae can only hardly be visualized in the
plant tissue.
The Department
of Molecular Phytopathology and Genetics (Prof. Schäfer) provided
us a GFP-expressing F. graminearum strain, which glows green when
irradiated with blue UV light. This makes it very easy to detect the fungus
on and inside the plant tissue. The infection course of F. graminearum
is observed in all layers of the caryopses, in isolated epicarps (Figure
5) and in cross sections of the caryopses (Figure 6).
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Figure 5. Hyphae of a GFP-expression strain of F. graminearum
in cells of the epicarp, i.e. the outmost layer of the barley caryopses.
The picture was taken 48 hours after artificial inoculation.
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Figure 6: Cross section of a barley caryopsis, 72 hours after artificial
infection with a GFP expressing F. graminearum strain. Green fluorescing
fungal mycelium is visible in the hypodermis.
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This project aims at the enlightenment of cellular and molecular processes
in the cereal-Fusarium interaction, to contribute to a solution
of the Fusarium problem.
English
literature:
Jansen
C, Schuphan I, Schmidt B (2000): Glufosinate metabolism in excised shoots
and leaves of twenty plant species. Weed Science 48 (3), 319-326.
Jansen
C, Korell M, Eckey C, Biedenkopf D, Kogel K-H (2001): Molecular analysis
of Mlg-mediated resistance and CIR in barely/powdery mildew-pathosystem:
II. Investigation of differential gene expression by SSH and RGA. Abstracts
International Workshop “Durable resistance in cereals – SAR and other strategies
to improve plant production”, Rauischholzhausen 29.03-01.04., 47.
Jansen
C, Stein E, Fensch R, Kogel K-H (2002): A genomic approach towards disease
resistance to Fusarium. 7th European Seminar on Fusarium-mycotoxins,
taxonomy and pathogenicity, 4.-7. Sep. 2002, Poznan, Poland; Book of abstracts,
p. 120.
Jansen
C, von Rüden S, Fensch R, Kogel K-H (2003): Consistency between degree
of susceptibility of barley root and spike tissue to Fusarium culmorum.
Mycotoxin
Research 19, 134-138
Eckey
C, Korell M, Leib K, Biedenkopf D, Jansen C, Kogel K-H (2004): Identification
of powdery mildew-induced barley genes by cDNA-AFLP: functional assessment
of an early expressed MAP kinase. Plant Molecular Biology 55: 1 - 15.
Jansen
C., Korell M., Eckey C., Biedenkopf D., Kogel K.-H.
(2005): Identification and transcriptional analysis of powdery mildew-induced
barley genes. Plant Science 168, 373-380.
Jansen,
C., Maier, F., Fensch, R., von Wettstein, D., Kogel, K.-H. und Schäfer,
W. (2004): Cytological analysis of the infection course of Fusarium graminearum
on barley caryopses. Proceedings of the 2nd International Symposium
on Fusarium Head Blight (2), p 459.
German literature:
Jansen C, Korell M, Eckey C, Kogel K-H (2000): Molekulare
Analyse im Gerste/Mehltau-Pathosystem: II Darstellung differentieller Genexpression
mittels SSH. Mitt. Biol. Bundesanst. Land- Forstwirtsch. 376, 394-395.
Jansen C, Eckey C, Korell M, Biedenkopf D, Micknass
U, Kogel K-H (2001): Molekulare Analyse der Mlg-vermittelten Resistenz
im Gerste/Mehltau-Pathosystem. Phytomedizin 31(2), 85-86.
Jansen C, Hückelhoven R, Fensch R, Schultheiß
H, Stein E, Dechert C, von Rüden S, Kogel K-H (2003): Strategien zur
Kontrolle von Fusarium in Getreide. Phytomedizin 33 (3), 28.
Jansen C, Kogel K-H (in press): GABI-Agrotec: Ein
nationales Verbundprojekt zur Sicherung der Nahrungsmittelqualität
von Getreideprodukten durch Grüne Gentechnik. Spiegel der Forschung,
Wissenschaftsmagazin der Justus-Liebig-Universität Gießen.
Jansen, C., Kogel K.-H.
(2004) GABI-Agrotec: Ein nationales Verbundprojekt zur Sicherung der Nahrungsmittelqualität
von Getreideprodukten durch Grüne Gentechnik. Spiegel der Forschung,
Wissenschaftsmagazin der Justus-Liebig-Universität Gießen, Jahrgang
21, Nr. ½ (ISSN 0176-3008).
Jansen, C., von Rüden, R., Fensch, R. und K.-H.
Kogel (2004) Isolierung und Charakterisierung Fusarium-responsiver Gene
der Gerste zur Identifizierung von Determinanten der Abwehr in Getreide.Mitt.
Biol. Bundesanst. Land- Forstwirtsch. 396, 283 f.
07.10.2005
