A class III peroxidase specifically expressed in pathogen-attacked barley epidermis contributes to basal resistance

Higher plants possess large multigene families encoding secreted class III peroxidase (Prx) proteins. In barley, two Prx cDNAs encoding HvPrx07 and HvPrx08 have been isolated and characterized to some extent with respect to a resistance-mediating function upon attack by the powdery-mildew fungus Blumeria graminis f.sp. hordei ( Bgh ). Here we present evidence for the tissue-specific accumulation of a new Prx mRNA, HvPrx40 , in Bgh -attacked epidermis of barley ( Hordeum vulgare ). The encoded protein is predicted to be secreted into the apoplastic space of epidermal cells due to the absence of a C-terminal extension, which distinguishes it from other Prx proteins reported to accumulate in leaf epidermis. Transient overexpression of HvPrx40 enhanced the resistance of wheat ( Triticum aestivum ) and barley against Blumeria graminis f.sp. tritici (wheat powdery mildew) and Bgh , respectively. These findings were complemented by transient-induced gene silencing showing hypersusceptibility of barley leaf epidermal cells to Bgh . The local accumulation of oxidized 3,3-diaminobenzidine that reflects H₂O₂ production at sites of attempted fungal penetration was not reduced in HvPrx40 -silenced cells, suggesting a role of this peroxidase other than the production of reactive oxygen species.     

Barley Rom1 antagonizes Rar1 function in Magnaporthe oryzae-infected leaves by enhancing epidermal and diminishing mesophyll defence

* Barley (Hordeum vulgare) is a host for Blumeria graminis f. sp. hordei (Bgh), which causes powdery mildew, and for the rice blast pathogen Magnaporthe oryzae. It has previously been shown that Rar1, initially identified in a mutational screen as being required for Mla12-specified Bgh-resistance, also controlled pathogenic growth of M. oryzae in barley. Here, we tested whether the rom1 mutation (restoration of Mla12-specified resistance), which restored resistance against Bgh in a susceptible rar1-2 genetic background, also influences the interaction between barley and M. oryzae. * Disease severity after infection with M. oryzae was analysed on rar1-2 mutants and rar1-2 rom1 double mutants. Microscopy and northern analysis were used to gain insight into cellular and molecular events. * On rar1-2 rom1 double mutant plants, the number of M. oryzae disease lesions was increased in comparison to the wild-type and the rar1-2 mutant which correlated with augmented epidermal penetration. However, a decrease in the lesion diameter, apparently conditioned in the mesophyll, was also observed. * These results highlight the impact of Rom1 in basal defence of barley against different pathogens. Importantly, a tissue-specific function for Rom1 with contrasting effects on epidermal and mesophyll defence was demonstrated.     

Superoxide and hydrogen peroxide play different roles in the nonhost interaction of barley and wheat with inappropriate formae speciales of Blumeria graminis

Nonhost resistance of cereals to inappropriate formae speciales of Blumeria graminis is little understood. However, on the microscopic level, nonhost defense to B. graminis is reminiscent of host defense preventing fungal development by penetration resistance and the hypersensitive cell death response (HR). We analyzed histochemically the accumulation of superoxide anion radicals (O2*-) and hydrogen peroxide (H2O2) at sites of B. graminis attack in nonhost barley and wheat. Superoxide visualized by subcellular reduction of nitroblue tetrazolium accumulated in association with successful fungal penetration in attacked cells and in cells neighboring HR. In contrast, H2O2 accumulated in cell wall appositions beneath fungal penetration attempts or in the entire epidermal cell during HR. The data provide evidence for different roles and sources of superoxide and H2O2 in the nonhost interaction of cereals with inappropriate formae speciales of B. graminis.     

The receptor-like MLO protein and the RAC/ROP family G-protein RACB modulate actin reorganization in barley attacked by the biotrophic powdery mildew fungus Blumeria graminis f.sp. hordei

Cytoskeleton remodelling is a crucial process in determining the polarity of dividing and growing plant cells, as well as during interactions with the environment. Nothing is currently known about the proteins, which regulate actin remodelling during interactions with invading pathogens. The biotrophic powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) invades susceptible barley (Hordeum vulgare L.) by penetrating epidermal cells, which remain intact during fungal development. In contrast, resistant host plants prevent infection by inhibiting penetration through apoplastic mechanisms, which require focusing defence reactions on the site of attack. We stained actin filaments in a susceptible Mlo-genotype and a near-isogenic race-non-specifically resistant barley mlo5-mutant genotype using fluorescence-labelled phalloidin after chemical fixation. This revealed that the actin cytoskeleton is differentially reorganized in susceptible and resistant hosts challenged by Bgh. Actin filaments were polarized towards the sites of attempted penetration in the resistant host, whereas when susceptible hosts were penetrated, a more subtle reorganization took place around fungal haustoria. Strong actin filament focusing towards sites of fungal attack was closely associated with successful prevention of penetration. Actin focusing was less frequent and seemingly delayed in susceptible wild-type barley expressing the susceptibility factor MLO. Additionally, single cell overexpression of a constitutively activated RAC/ROP G-protein, CA RACB, another potential host susceptibility factor and hypothetical actin cytoskeleton regulator, partly inhibited actin reorganization when under attack from Bgh, whereas knockdown of RACB promoted actin focusing. We conclude that RACB and, potentially, MLO are host proteins involved in the modulation of actin reorganization and cell polarity in the interaction of barley with Bgh.     

Tissue-Specific Expression of Germin-Like Oxalate Oxidase during Development and Fungal Infection of Barley Seedlings

Oxalate oxidase activity was detected in situ during the development of barley seedlings. The presence of germin-like oxalate oxidase was confirmed by immunoblotting using an antibody directed against wheat germin produced in Escherichia coli, which is shown to cross-react with barley (Hordeum vulgare) oxalate oxidase and by enzymatic assay after electrophoresis of the protein extracts on polyacrylamide gels. In 3-d-old barley seedlings, oxalate oxidase is localized in the epidermal cells of the mature region of primary roots and in the coleorhiza. After 10 d of growth, the activity is detectable only in the coleorhiza. Moreover, we show that oxalate oxidase is induced in barley leaves during infection by the fungus Erysiphe graminis f. sp. hordei but not by wounding. Thus, oxalate oxidase is a new class of proteins that responds to pathogen attack. We propose that oxalate oxidase could have a role in plant defense through the production of H2O2.     

The PEN1 syntaxin defines a novel cellular compartment upon fungal attack and is required for the timely assembly of papillae

Attack by the host powdery mildew Erysiphe cichoracearum usually results in successful penetration and rapid proliferation of the fungus on Arabidopsis. By contrast, the nonhost barley powdery mildew Blumeria graminis f. sp. hordei (Bgh) typically fails to penetrate Arabidopsis epidermal cells. In both instances the plant secretes cell wall appositions or papillae beneath the penetration peg of the fungus. Genetic screens for mutations that result in increased penetration of Bgh on Arabidopsis have recently identified the PEN1 syntaxin. Here we examine the role of PEN1 and of its closest homologue, SYP122, identified as a syntaxin whose expression is responsive to infection. pen1 syp122 double mutants are both dwarfed and necrotic, suggesting that the two syntaxins have overlapping functions. Although syp122-1 and the cell wall mur mutants have considerably more pronounced primary cell wall defects than pen1 mutants, these have relatively subtle or no effects on penetration resistance. Upon fungal attack, PEN1 appears to be actively recruited to papillae, and there is a 2-h delay in papillae formation in the pen1-1 mutant. We conclude that SYP122 may have a general function in secretion, including a role in cell wall deposition. By contrast, PEN1 appears to have a basal function in secretion and a specialized defense-related function, being required for the polarized secretion events that give rise to papilla formation.     

Host and non-host pathogens elicit different jasmonate/ethylene responses in Arabidopsis

Arabidopsis does not support the growth and asexual reproduction of the barley pathogen, Blumeria graminis f. sp. hordei Bgh). A majority of germlings fail to penetrate the epidermal cell wall and papillae. To gain additional insight into this interaction, we determined whether the salicylic acid (SA) or jasmonate (JA)/ethylene (ET) defence pathways played a role in blocking barley powdery mildew infections. Only the eds1 mutant and NahG transgenics supported a modest increase in penetration success by the barley powdery mildew. We also compared the global gene expression patterns of Arabidopsis inoculated with the non-host barley powdery mildew to those inoculated with a virulent, host powdery mildew, Erysiphe cichoracearum. Genes repressed by inoculations with non-host and host powdery mildews relative to non-inoculated control plants accounted for two-thirds of the differentially expressed genes. A majority of these genes encoded components of photosynthesis and general metabolism. Consistent with this observation, Arabidopsis growth was inhibited following inoculation with Bgh, suggesting a shift in resource allocation from growth to defence. A number of defence-associated genes were induced during both interactions. These genes likely are components of basal defence responses, which do not effectively block host powdery mildew infections. In addition, genes encoding defensins, anti-microbial peptides whose expression is under the control of the JA/ET signalling pathway, were induced exclusively by non-host pathogens. Ectopic activation of JA/ET signalling protected Arabidopsis against two biotrophic host pathogens. Taken together, these data suggest that biotrophic host pathogens must either suppress or fail to elicit the JA/ET signal transduction pathway.     

Barley RIC171 interacts with RACB in planta and supports entry of the powdery mildew fungus

RHO-like GTPases of plants (ROPs, also called RACs) are involved in plant development and interaction with the environment. The barley ROP protein RACB is involved in susceptibility to the fungal pathogen Blumeria graminis f.sp. hordei ( Bgh ) . By screening barley sequence databases for potential protein interactors of plant RHO-like proteins, we identified a ROP-interactive CRIB (CDC42/RAC interactive binding) motif containing protein of 171 amino acids (RIC171). The protein interacted with constitutively activated RACB in a targeted yeast two-hybrid assay. By use of split yellow fluorescing protein fusions, we demonstrated that RIC171 interacts with constitutively activated (CA) RACB-G15V but not with dominant negative RACB-T20N in planta . Transient overexpression of RIC171, similar to overexpression of CA RACB-G15V, rendered epidermal cells more susceptible to penetration by Bgh . In contrast, expression of a 46-amino-acid RIC171-CRIB peptide, which was sufficient to interact with CA RACB-G15V, had a dominant negative effect and reduced susceptibility to Bgh . A red fluorescing DsRED–RIC171 fusion protein colocalized with green fluorescing GFP–RACB-G15V at the cell periphery. Coexpression with CA RACB-G15V but not with RACB-T20N increased peripheral localization of DsRED–RIC171. Additionally, DsRED–RIC171 accumulated at sites of fungal attack, suggesting enhanced ROP activity at sites of attempted fungal penetration.     

Multivesicular bodies participate in a cell wall-associated defence response in barley leaves attacked by the pathogenic powdery mildew fungus

Localized cell wall modification and accumulation of antimicrobial compounds beneath sites of fungal attack are common mechanisms for plant resistance to fungal penetration. In barley (Hordeum vulgare) leaves, light-microscopically visible vesicle-like bodies (VLBs) containing H(2)O(2) or phenolics frequently accumulate around cell wall appositions (syn. papillae), in which the penetration attempt of the biotrophic powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) is halted. By ultrastructural analyses, we demonstrated that the Bgh-induced VLBs represent different structures. VLBs intensively stained by H(2)O(2)-reactive dyes were actually small papillae instead of cytoplasmic vesicles. Other VLBs were identified as osmiophilic bodies or multivesicular compartments, designated paramural bodies (PMBs) and multivesicular bodies (MVBs). MVBs seemingly followed two distinct pathways: either they were engulfed by the tonoplast for degradation in the vacuole or they fused with the plasma membrane to release their internal vesicles into the paramural space and hence could be the origin of PMBs. MVBs and PMBs appeared to be multicomponent kits possibly containing building blocks to be readily assembled into papilla and antimicrobial compounds to be discharged against fungal penetration. Finally, we propose that released paramural vesicles might be similar to exosomes in animal cells.     

Functional analysis of barley RAC/ROP G-protein family members in susceptibility to the powdery mildew fungus

Small 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 Ca2+, 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 identified 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-specific 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 fluorescent 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.     

The barley apoptosis suppressor homologue BAX inhibitor-1 compromises nonhost penetration resistance of barley to the inappropriate pathogen Blumeria graminis f. sp. tritici

BAX inhibitor-1 (BI-1) proteins have been characterized as suppressors of programmed cell death in mammals and plants. The barley BI-1 is a suppressor of nonspecific background resistance and mlo-mediated penetration resistance to the biotrophic fungal pathogen Blumeria graminis f. sp. hordei when overexpressed in epidermal cells of barley. We report here that BI-1 expression is also slightly up-regulated during interaction with the inappropriate wheat pathogen Blumeria graminis f. sp. tritici. Significantly, overexpression of BI-1 in single epidermal cells of barley by microprojectile-mediated transformation rendered cells susceptible to penetration by inappropriate B. graminis f. sp. tritici. The degree of transgene-induced accessibility to B. graminis f. sp. tritici was thereby similar to the effect achieved by overexpression of the defense suppressor gene Mlo and could not be further enhanced by double expression of both BI-1 and Mlo. Confocal laser scanning microscopy was used to locate a functional green fluorescing GFP:BI-1 fusion protein in endomembranes and the nuclear envelope of barley epidermal cells. Together, enhanced expression of barley BI-1 suppresses penetration resistance to B. graminis f. sp. tritici, linking barley nonhost resistance with cell death regulation.     

Stomatal lock-open, a consequence of epidermal cell death, follows transient suppression of stomatal opening in barley attacked by Blumeria graminis

Blumeria graminis f.sp. hordei (Bgh) attack disrupted stomatal behaviour, and hence leaf water conductance (g(l)), in barley genotypes Pallas and Ris?-S (susceptible), P01 (with Mla1 conditioning a hypersensitive response; HR), and P22 and Ris?-R (with mlo5 conditioning papilla-based penetration resistance). Inoculation caused some stomatal closure well before the fungus attempted infection. Coinciding with epidermal cell penetration, stomatal opening in light was also impeded, although stomata of susceptible and mlo5 lines remained largely able to close in darkness. Following infection, in susceptible lines stomata closed in darkness but opening in light was persistently impeded. In Ris?-R, stomata recovered nearly complete function by approximately 30 h after inoculation, i.e. after penetration resistance was accomplished. In P01, stomata became locked open and unable to close in darkness shortly after epidermal cells died due to HR. In the P22 background, mlo5 penetration resistance was often followed by consequential death of attacked cells, and here too stomata became locked open, but not until approximately 24 h after pathogen attack had ceased. The influence of epidermal cell death was localized, and only affected stomata within one or two cells distance. These stomata were unable to close not only in darkness but also after application of abscisic acid and in wilted leaves suffering drought. Thus, resistance to Bgh based on HR or associated with cell death may have previously unsuspected negative consequences for the physiological health of apparently 'disease-free' plants. The results are discussed in relation to the control of stomatal aperture in barley by epidermal cells.