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