Induced Resistance (IR) in Barley
Induzierte Resistenz in Getreide

Group leader: Dr. rer. nat. Gregor Langen
Coworkers: Dr. rer. nat. Ingo Ciolkowski, Martina Claar, Dipl. Biol. Bettina Kah, Dipl. Biol. Katja Leib, Elke Stein, Petra Theuer

control, non-induced

after pre-treatment with resistance inducer DCINA

Powdery mildew pustule formation on a susceptible barley cultivar
Blumeria graminis f.sp. hordei causes powdery mildew on barley. The biotrophic ectoparasite grows on the leaf surface invading the epidermal cells only with its feeding organ, the haustorium. On susceptible barley cultivars a visible white mycelium is produced. Pre-treatment of susceptible barley plants with a resistance inducer such as DCINA decreases the number of pustules (successful infections).

Focus of our research is on inducible defence responses in cereals like barley. Susceptible barley plants are able to trigger the same types of defence responses like genetically resistant lines but only after a pre-treatment with an inducer (priming). Studies in dicot plants like Arabidopsis thaliana revealed that involved signals and mechanisms are similar to the innate immunity system in animal cells. Resistance can be induced locally (LAR) and systemically (SAR, systemic acquired resistance) by an avirulent pathogen or a chemical. Induced resistance was shown to be effective against a broad spectrum of pathogens (viruses, bacteria, and fungi). 

In barley, the same types of chemicals as in Arabidopsis and tobacco are effective in inducing resistance against powdery mildew like salicylic acid (SA), dichloroisonicotinic acid (DCINA) and acibenzolar-S-methyl (ASM, = benzothiadiazole, BTH). Induced defences against the biotrophic fungus include production of antifungal proteins, cell wall appositions (papillae) and cell death (hypersensitive reaction) involving reactive oxygen species. In chemically induced and genetically resistant lines the microscopic observable reactions are similar.

Aim of our investigations is the characterisation of the IR mechanism in barley. 

papilla	hypersensitive cell death (HR)

A fungal spore of the powdery mildew fungus Blumeria graminis f.sp. hordei has germinated on the surface of a barley leaf (A). The appressorium at the end of the germ tube produced a penetration peg in an attempt to enter the epidermal cell which is indispensable for the biotrophic fungus to produce its feeding organ, the haustorium. A cell wall apposition (papilla) was formed by the plant cell in response to this attack preventing the fungus from penetration. Additionally to or instead of papilla formation the accumulation of autofluorescing material is often detectable under UV light (B) indicative of the hypersensitive response (HR) / cell death reaction of the attacked cell which also stops the fungus.

Signalling pathways in defence responses: Gene regulation

Although the same types of chemicals as in dicots are effective in barley, we found a poor correlation regarding gene induction of marker genes for SAR in tobacco and Arabidopsis. Applying suppressive subtraction hybridisation we identified 9 Bci (barley chemically induced) genes. Three of them are very specifically induced by IR inducing chemicals but not by various tested pathogens. 

Bci4 transcript accumulation in barley after treatment with several resistance inducers

Function of the Bci genes in resistance against powdery mildew is being tested in transient transformations by particle bombardment of epidermal cells.

Transiently transformed barley epidermal cell expressing the marker gene GUS visible as turquoise staining. The powdery mildew fungus was stained with blue ink. The fungus was able to establish a functional haustorium in the cell and started to grow on the surface of the leaf (compatible interaction).

In order to identify more genes differentially regulated during IR in barley we established a macroarray with appr. 1500 cDNAs of BTH treated barley epidermis. These filters are being hybridised with complex cDNA probes of induced and mock treated plants to identify differentially regulated genes to get an insight into transcriptome change during IR in barley.

Barley epidermal cDNA filter. Each cDNA was spotted twice. Filters are hybridised with complex cDNA probes of induced and mock treated plants.

Signalling pathways in defence responses: promoter studies

We are interested in gene regulation during IR. For some of the Bci genes we isolated the 5’-upstream genomic sequence containing the putative promoters with regulatory elements. The promoters are being analysed in transient assays to identify IR responsive motifs.

Transgenic plants

First transgenic barley and wheat plants were produced overexpressing Bci genes or bearing Bci promoter::reporter constructs. Recently, we established an own transformation group in the department (see Imani’s group).

GFP (green fluorescence protein) expressed in barley epidermal and mesophyll cells (red: chloroplasts)

WRKY transcription factors in barley

WRKY transcription factors form a large plant specific gene family (about 77 members in rice). They are able to bind to W-boxes, which are overrepresented in the promoters of genes related to pathogen resistance and senescence. 

During a cDNA-AFLP assay we discovered a WRKY factor upregulated after Bgh inoculation. Until now we managed to characterise at least three HvWRKYs in terms of expression patterns. Transient transformation assays revealed their importance in the interaction of barley with Bgh. Transgenic barley plants are being generated to affirm these results. 

HvWRKY3:GFP fusion protein transiently expressed in barley epidermal cells. The fusion-protein (green) is localized in the nucleus (red: DsRed marking transformed cells).

Members of the team

Ingo Ciolkowski (Dr. rer. nat.)

Martina Claar (Technical Assistant)

Bettina Kah (Dipl. Biol., PhD student)

Katja Leib (Dipl. Biol., PhD student)

Elke Stein (Technical Assistant)

Petra Theuer (Technical Assistant)

Alumni

Katrin Beßer (Dr. rer. nat.)

Uta Geldermann (Dr. agr.)

Sanjay Kumar Jain (Dr. agr.)

Publications

Langen G, Imani J, Altincicek B, Kieseritzky G, Kogel KH, Vilcinskas A. (2006) Expression of gallerimycin, a novel antifungal insect defensin from the greater wax moth Galleria mellonella, confers resistance to pathogenic fungi in tobacco. Biological Chemistry, 387 (5), 549-557 2006.

Eichmann R, Biemelt S, Schäfer P, Scholz U, Jansen C, Felk A, Schäfer W, Langen G, Sonnewald U, Kogel KH, Hückelhoven R. Macroarray expression analysis of barley host susceptibility and non-host resistance to Blumeria graminis. J. Plant Physiology 163 (6), 657-670, 2006.

Kogel KH and Langen G. Induced Disease Resistance and Gene Expression in Cereals. Cellular Microbiology 7 (11), 1555-1564, 2005.

Eckey C, Korell M, Leib K, Biedenkopf D, Jansen C, Langen G, and Kogel KH. Identification of powdery mildew-induced barley genes by cDNA-AFLP: Functional assessment of an early expressed MAP kinase. Plant Mol. Biol. 55, 1-15, 2004.

Jain SK, Langen G, Hess W, Börner T, Hückelhoven R, KH Kogel. The White Barley Mutant Albostrians Shows Enhanced Resistance to the Biotroph Blumeria graminis f. sp. hordei. Mol. Plant-Microbe Interact. 17, (4), 374–382, 2004. 

Mouhanna AM, Nasrallah A, Langen G, Schlösser E. Surveys for Beet necrotic yellow vein virus (the Cause of Rhizomania), other viruses, and Soil-borne Fungi Infecting Sugar Beet in Syria. J. of Phytopathology. 150, 657-662, 2002.

Geldermann U, Langen G, Kogel KH. Promotor studies of chemically induced Bci-genes in the pathosystem barley – powdery mildew. Plant Protect. Sci. 38 (Special Issue 2), 487 - 489, 2002.

Kumar J, Schäfer P, Hückelhoven R, Langen G, Baltruschat H, Stein E, Nagarajan S, Kogel KH. Bipolaris sorokiniana, a cereal pathogen of global concern: cytological and molecular approaches towards better control. Mol. Plant Path. 3(4), 185-195, 2002

Langen G, Beßer K, Eichmann R, Geldermann U, Kah B, von Rüden S, Kogel KH. BTH induced barley genes: Co-regulated or directly involved in powdery mildew resistance? Plant Protect. Sci. 38 (Special Issue 1), 243, 2002.

Schäfer P, Hückelhoven R, Langen G, Kumar J, Kogel KH. Bipolaris sorokiniana on cereals: a new model for molecular work on hemibiotrophy. Plant Protect. Sci. 38 (Special Issue 1), 245, 2002.

Imani J, Tran Thi L, Langen G, Arnholdt-Schmitt B, Roy S, Lein C, Kumar A, Neumann KH. Somatic embryogenesis and DNA organization of genomes from selected Daucus species. Plant Cell Reports 20 (6), 537-541, 2001.

Kumar J, Langen G, Stein E, Kogel KH. Chemically induced resistance in monocots with emphasis on barley. In: Nagarajan S, Singh DP (eds.), Role of Resistance in Intensive Agriculture, Kalyani Publishers, Ludhiana, India, 216–222, 2001.

Beßer K, Jarosch B, Langen G, Kogel KH. Expression analysis of genes induced in barley after chemical activation reveals distinct disease resistance pathways. Mol. Plant Path. 1 (5), 277-286, 2000.

Langen G, Beßer K, Kogel KH. Identification and expression analysis of genes induced in barley after chemical activation of disease resistance. Acta Phytopath. et Entomol. Hungarica 35 (1-4), 1, 2000.

Langen G, Beßer K, Jarosch B, Düringer M, Stein E, Kogel KH. Identifizierung neuer Gerstengene mit breiter Wirkung auf Krankheitsresistenzen. In: Kogel KH et al., Erhöhung der Krankheitsresistenz von Getreiden. Spiegel der Forschung 17 No. 2, 21 - 30, 2000.

Kogel, KH, Beckhove U, Jarosch B, Hückelhoven R, Schiffer R, Beßer K, Langen G, Korell M. Die Pflanze wehrt sich selbst - Resistenzaktivierung in Kulturpflanzen. Spiegel der Forschung 15, 54 - 61, 1998.

Langen G, Hückelhoven R, Beßer K, Schaffrath U, Kogel KH. Eine vorläufige Bewertung der Rolle reaktiver Sauerstoffspezies bei Abwehrreaktionen von Getreide gegenüber pilzlichen Pathogenen. Gesunde Pflanzen 50, 196 - 202, 1998.

Langen G, Beißmann B, Reisener HJ, Kogel KH. A ß-1,3-D endo-mannanase from culture filtrates of the hyperparasites Verticillium lecanii and Aphanocladium album that specifically lyses the germ pore plug from uredospores of Puccinia graminis f. sp. tritici. Can. J. Bot. 70, 853 - 860, 1992.

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