SummarySmall monomeric G-proteins of the plant ras (rat sarcome oncogene product) related C3 botulinum toxin substrate (RAC)/Rho of plants (ROP) family are molecular switches in signal transduction of many cellular processes. RAC/ROPs regulate hormone effects, subcellular gradients of Ca 2 , the organisation of the actin cytoskeleton and the production of reactive oxygen intermediates. Therefore, we followed a genetic bottom-up strategy to study the role of these proteins during the interaction of barley (Hordeum vulgare L.) with the fungal biotrophic pathogen Blumeria graminis f.sp. hordei (Bgh). We identi®ed six barley RAC/ ROP proteins and studied their gene expression. Five out of six Rac/Rop genes were expressed constitutively in the leaf epidermis, which is the site of interaction with Bgh. None of the genes showed enhancement of mRNA abundance after inoculation with Bgh. After microprojectile mediated transformation of single barley epidermal cells with constitutively activated mutant RAC/ROP proteins, we found an RAC/ ROP-speci®c enhancement of pathogen accessibility, tagging HvRACB, HvRAC3 and HvROP6 as host proteins potentially involved in the establishment of susceptibility to Bgh. Confocal laser scanning microscopy (CLSM) of green¯uorescent protein (GFP):HvRAC/ROP-transformed cells revealed varying strengths of plasma membrane association of barley RAC/ROPs. The C-terminal CAAX motif for presumable prenylation or the C-terminal hypervariable region (HVR), respectively, were required for membrane association of the RAC/ROPs. Proper intracellular localisation was essential for HvRACB and HvRAC3 function. Together, our data support the view that different paths of host signal transduction via RAC/ROP G-proteins are involved in processes supporting parasitic entry into epidermal host cells.

Cell death regulation is linked to pathogen defense in plants and animals. Execution of apoptosis as one type of programmed cell death in animals is irreversibly triggered by cytochrome c release from mitochondria via pores formed by BAX proteins. This type of programmed cell death can be prevented by expression of BAX inhibitor 1 (BI-1), a membrane protein that protects cells from the effects of BAX by an unknown mechanism. In barley, a homologue of the mammalian BI-1 is expressed in response to inoculation with the barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh). We found differential expression of BI-1 in response to Bgh in susceptible and resistant plants. Chemical induction of resistance to Bgh by soil drench treatment with 2,6-dichloroisonicotinic acid led to down-regulation of the expression level of BI-1. Importantly, single-cell transient overexpression of BI-1 in epidermal leaf tissue of susceptible barley cultivar Ingrid led to enhanced accessibility, resulting in a higher penetration efficiency of Bgh on BI-1-transformed cells. In Bgh-resistant mlo5 genotypes, which do not express the negative regulator of defense and cell death MLO, overexpression of BI-1 almost completely reconstituted susceptibility to fungal penetration. We suggest that BI-1 is a regulator of cellular defense in barley sufficient to substitute for MLO function in accessibility to fungal parasites.

Small GTP-binding proteins such as those from the RAC family are cytosolic signal transduction proteins that often are involved in processing of extracellular stimuli. Plant RAC proteins are implicated in regulation of plant cell architecture, secondary wall formation, meristem signaling, and defense against pathogens. We isolated a RacB homolog from barley (Hordeum vulgare) to study its role in resistance to the barley powdery mildew fungus (Blumeria graminis f.sp. hordei). RacB was constitutively expressed in the barley epidermis and its expression level was not strongly influenced by inoculation with B. graminis. However, after biolistic bombardment of barley leaf segments with RacB-double-stranded RNA, sequencespecific RNA interference with RacB function inhibited fungal haustorium establishment in a cell-autonomous and genotype-specific manner. Mutants compromised in function of the Mlo wild-type gene and the Ror1 gene (genotype mlo5 ror1) that are moderately susceptible to B. graminis showed no alteration in powdery mildew resistance upon RacB-specific RNA interference. Thus, the phenotype, induced by RacB-specific RNA interference, was apparently dependent on the same processes as mlo5-mediated broad resistance, which is suppressed by ror1. We conclude that an RAC small GTP-binding protein is required for successful fungal haustorium establishment and that this function may be linked to MLO-associated functions.Complete resistance of barley (Hordeum vulgare) to the biotrophic, fungal pathogen Blumeria graminis f.sp. hordei (Bgh) is mediated by major resistance genes such as the Mla genes or by loss of MLO function in Mlo-mutant genotypes such as mlo5-barley (Jørgensen, 1994;Schulze-Lefert and Vogel, 2000). The latter is expressed exclusively via penetration resistance, which is characterized by formation of cell wall appositions and accumulation of phytoalexins, pathogenesis-related gene transcripts, and hydrogen peroxide (Stolzenburg et al., 1984;Zeyen et al., 1993; Peterhä nsel et al., 1997;von Rö penack et al., 1998;Hü ckelhoven et al., 1999Hü ckelhoven et al., , 2000b. All of these characteristics are also found in susceptible barley, albeit to a lower extent, meaning that the mlo alleles confer a primed responsiveness for these defense reactions or the functional MLO is a control element of these fundamental resistance mechanisms (Bü schges et al., 1997; Peterhä nsel et al., 1997).It is intriguing that Bgh-resistant mlo genotypes show hypersusceptibility to Magnaporthe grisea and to toxic culture filtrates of Cochliobolus sativus (Jarosch et al., 1999;Kumar et al., 2001). Thus, Mlo exerts an ambivalent role in controlling resistance to the biotroph Bgh and susceptibility to the hemibiotroph M. grisea. The MLO protein is a membrane-spanning protein reminiscent of a G-protein coupled receptor (Devoto et al., 1999). In animals, such proteins interact with heterotrimeric G-proteins and/or small GTP-binding proteins via different cytoplasmic domains (Naor et al., 2000). Small GTP-binding proteins such as ...

Summary In barley, non-host resistance against the wheat powdery mildew fungus (Blumeria graminis f.sp. tritici, Bgt) is associated with the formation of cell wall appositions and a hypersensitive reaction in which epidermal cells die rapidly in response to fungal attack. In the interaction of barley with the pathogenic barley powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh), these defence reactions are also associated with accumulation of H(2)O(2). To elucidate the mechanism of non-host resistance, the accumulation of H(2)O(2) in response to Bgt was studied in situ by histochemical staining with diaminobenzidine. H(2)O(2) accumulation was found in cell wall appositions under appressoria from Bgt and in cells undergoing a hypersensitive reaction. A mutation (mlo5) at the barley Mlo locus, that confers broad spectrum resistance to Bgh, did not influence the barley defence phenotype to Bgt. Significantly, Bgt triggered cell death on mlo5-barley while Bgh did not.

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