RHO-like monomeric G-proteins of plants (ROPs, also called RACs), are involved in plant development and interaction with the environment. The barley (Hordeum vulgare) ROP protein HvRACB has been shown to be required for entry of the biotrophic powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) into living host cells. To get a deeper insight into evolutionarily conserved functions of ROPs in cell polarity and pathogen responses, we stably expressed constitutively activated (CA) mutant variants of different barley ROPs (HvRACB, HvRAC1, HvRAC3) in barley. CA HvROPs induced epidermal cell expansion and/or abolished polarity in tip growing root hairs. All three CA HvROPs enhanced susceptibility of barley to penetration by Bgh whereas only CA HvRAC1 supported whole cell H(2)O(2) production in non-penetrated cells. Despite increasing penetration by Bgh, CA HvRAC1 promoted callose deposition at sites of fungal attack and resistance to penetration by Magnaporthe oryzae. The data show an involvement of ROPs in polar growth processes of the monocot barley and in responses to fungal pathogens with different life style.

Plant respiratory burst oxidase homologs are prominent sources of reactive oxygen species (ROS) in signal transduction and in interaction with microbes. However, the function of respiratory burst oxidase homologue (RBOH) genes in interaction with microbes might differ for certain plant and pathogen species. We produced transgenic barley knock down (KD) for the HvRBOHF2 isoform of NADPH oxidases. Young HvRBOHF2 KD shoots did not show obvious morphological alterations from the wild type but adult HvRBOHF2 KD plants developed fewer tillers, were less fertile, and showed spontaneous cell death in leaf mesophyll. Additionally, HvRBOHF2 KD plants were unable to contain wound-induced cell death. Before developmental failure became obvious, young HvRBOHF2 KD seedlings were much more susceptible to penetration by the biotrophic powdery mildew fungus Blumeria graminis f. sp. hordei. Strikingly, the B. graminis f. sp. hordei-induced cell-wall-associated oxidative burst was not substantially attenuated in HvRBOHF2 KD plants but enhanced susceptibility apparently influenced the subcellular site of hydrogen peroxide accumulation. Taken together, misexpression of HvRBOHF2 caused failure of barley to normally develop penetration resistance to B. graminis f. sp. hordei and to control leaf cell death.

Plant primary energy metabolism is profoundly reorganized under biotic stress conditions and there is increasing evidence for a role for the fermentative pathway in biotic interactions. However, the mechanisms regulating metabolic reprogramming are not well understood despite its critical function in the biotic stress response. Here the function of alcohol dehydrogenase (ADH) in the interaction of barley with the parasitic fungus Blumeria graminis f.sp. hordei (Bgh) is addressed. Challenge of susceptible barley leaves with Bgh resulted in transcriptional activation of HvADH1 and an induction of ADH enzyme activity starting 24 h after infection and reaching a clear-cut effect 4 d after infection. This increase in ADH enzyme activity was not observed in the resistant near-isogenic mlo5 line. Moreover, an induction of ADH enzyme activity by Bgh was enhanced in the presence of sucrose in hydroponically grown seedlings. Transient knock-down or overexpression of HvADH1 in barley epidermal cells mediated a decrease or increase in the penetration success of Bgh, respectively. Inhibition of ADH activity by pyrazole resulted in a delay in symptoms. The pyrazole effect could be overcome by adding glucose to the incubation medium, pinpointing a nutritional effect of ADH in the barley–Bgh interaction. Taken together, misexpression of pathogen-inducible HvADH1 or variation of ADH activity modulates the pathogen response of barley to the biotrophic fungal parasite Bgh. In this way, ADH knock-down/inhibition results in reduced fungal success. The possibility is discussed that ADH activity supports biotrophy by maintaining glycolytic metabolism in pathogen-stressed barley.

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