The germinlike protein GLP4 exhibits superoxide dismutase activity and is an important component of quantitative resistance in wheat and barley

Germinlike proteins (GLP) are encoded in plants by a gene family with proposed functions in plant development and defense. Genes of GLP subfamily 4 of barley (HvGLP4, formerly referred to as HvOxOLP) and the wheat orthologue TaGLP4 (formerly referred to as TaGLP2a) were previously found to be expressed in pathogen-attacked epidermal tissue of barley and wheat leaves, and the corresponding proteins are proposed to accumulate in the apoplast. Here, the role of HvGLP4 and TaGLP4 in the defense of barley and wheat against Blumeria graminis (DC.) E. O. Speer, the cereal powdery mildew fungus, was examined in an epidermal transient expression system and in transgenic Arabidopsis thaliana plants overexpressing His-tagged HvGLP4. Leaf extracts of transgenic Arabidopsis overexpressing HvGLP4 contained a novel His-tagged protein with superoxide dismutase activity and HvGLP4 epitopes. Transient overexpression of TaGLP4 and HvGLP4 enhanced resistance against B. graminis in wheat and barley, whereas transient silencing by RNA interference reduced basal resistance in both cereals. The effect of GLP4 overexpression or silencing was strongly influenced by the genotype of the plant. The data suggest that members of GLP subfamily 4 are components of quantitative resistance in both barley and wheat, acting together with other, as yet unknown, plant components.     

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.     

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 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.     

Arabidopsis nonhost resistance gene PSS1 confers immunity against an oomycete and a fungal pathogen but not a bacterial pathogen that cause diseases in soybean

ABSTRACT: BACKGROUND: Nonhost resistance (NHR) provides immunity to all members of a plant species against all isolates of a microorganism that is pathogenic to other plant species. Three Arabidopsis thaliana PEN (penetration deficient) genes, PEN1, 2 and 3 have been shown to provide NHR against the barley pathogen Blumeria graminis f. sp. hordei at the prehaustorial level. Arabidopsis pen1-1 mutant lacking the PEN1 gene is penetrated by the hemibiotrophic oomycete pathogen Phytophthora sojae, the causal organism of the root and stem rot disease in soybean. We investigated if there is any novel nonhost resistance mechanism in Arabidopsis against the soybean pathogen, P. sojae. RESULTS: The P. sojae susceptible (pss) 1 mutant was identified by screening a mutant population created in the Arabidopsis pen1-1 mutant that lacks penetration resistance against the non adapted barley biotrophic fungal pathogen, Blumeria graminis f. sp. hordei. Segregation data suggested that PEN1 is not epistatic to PSS1. Responses of pss1 and pen1-1 to P. sojae invasion were distinct and suggest that PSS1 may act at both pre- and post-haustorial levels, while PEN1 acts at the pre-haustorial level against this soybean pathogen. Therefore, PSS1 encodes a new form of nonhost resistance. The pss1 mutant is also infected by the necrotrophic fungal pathogen, Fusarium virguliforme, which causes sudden death syndrome in soybean. Thus, a common NHR mechanism is operative in Arabidopsis against both hemibiotrophic oomycetes and necrotrophic fungal pathogens that are pathogenic to soybean. However, PSS1 does not play any role in immunity against the bacterial pathogen, Pseudomonas syringae pv. glycinea, that causes bacterial blight in soybean. We mapped PSS1 to a region very close to the southern telomere of chromosome 3 that carries no known disease resistance genes. CONCLUSIONS: The study revealed that Arabidopsis PSS1 is a novel nonhost resistance gene that confers a new form of nonhost resistance against both a hemibiotrophic oomycete pathogen, P. sojae and a necrotrophic fungal pathogen, F. virguliforme that cause diseases in soybean. However, this gene does not play any role in the immunity of Arabidopsis to the bacterial pathogen, P. syringae pv. glycinea, which causes bacterial blight in soybean. Identification and further characterization of the PSS1 gene would provide further insights into a new form of nonhost resistance in Arabidopsis, which could be utilized in improving resistance of soybean to two serious pathogens.     

Transient expression of members of the germin-like gene family in epidermal cells of wheat confers disease resistance

The wheat genome encodes a family of germin-like proteins that differ with respect to regulation and tissue specificity of expression of the corresponding genes. While germin exhibits oxalate oxidase (E.C. 1.2.3.4.) activity, the germin-like proteins (GLPs) have no known enzymatic activity. A role of oxalate oxidase in plant defence has been proposed, based on the capacity of the enzyme to produce H2O2, a reactive oxygen species. The role in defence of germin and other members of the germin-like gene family was functionally assessed in a transient assay system based on particle bombardment of wheat leaves. Transient expression of the pathogen-induced germin gf-2.8 gene, but not of the constitutively expressed HvGLP1 gene, reduced the penetration efficiency of Blumeria (syn. Erysiphe) graminis f.sp. tritici, the causal agent of wheat powdery mildew, on transformed cells. Two engineered germin-gf-2.8 genes and the TaGLP2a gene, which all encoded proteins without oxalate oxidase activity, also reduced the penetration efficiency of the fungus, demonstrating that oxalate oxidase activity is not required for conferring enhanced resistance. Instead, activity tagging experiments showed that in cells transiently expressing the germin gf-2.8 gene, the transgene product became insolubilized at sites of attempted fungal penetration where localised production of H2O2 was observed. Thus, germin and GLPs may play a structural role in cell-wall re-enforcement during pathogen attack.     

The Arabidopsis thaliana ABC protein superfamily, a complete inventory

We describe the first complete inventory of ATP-binding cassette (ABC) proteins from a multicellular organism, the model plant Arabidopsis thaliana. By the application of several search criteria, Arabidopsis was found to contain a total of 129 open reading frames (ORFs) capable of encoding ABC proteins, of which 103 possessed contiguous transmembrane spans and were identified as putative intrinsic membrane proteins. Fifty-two of the putative intrinsic membrane proteins contained at least two transmembrane domains (TMDs) and two nucleotide-binding folds (NBFs) and could be classified as belonging to one of five subfamilies of full-molecule transporters. The other 51 putative membrane proteins, all of which were half-molecule transporters, fell into five subfamilies. Of the remaining ORFs identified, all of which encoded proteins lacking TMDs, 11 could be classified into three subfamilies. There were no obvious homologs in other organisms for 15 of the ORFs which encoded a heterogeneous group of non-intrinsic ABC proteins (NAPs). Unrooted phylogenetic analyses substantiated the subfamily designations. Notable features of the Arabidopsis ABC superfamily was the presence of a large yeast-like PDR subfamily, and the absence of genes encoding bona fide cystic fibrosis transmembrane conductance regulator (CFTR), sulfonylurea receptor (SUR), and heavy metal tolerance factor 1 (HMT1) homologs. Arabidopsis was unusual in its large allocation of ORFs (a minimum of 0.5%) to members of the ABC protein superfamily.     

Stable expression of a defense-related gene in wheat epidermis under transcriptional control of a novel promoter confers pathogen resistance

Tissue-specific or regulated expression of transgenes is desirable in order to prevent pleiotropic side effects of putatively harmful transgene products as well as loss of energy resources due to unnecessary accumulation of transgene products. Epidermis-specific expression would be useful for many defense-related genes directed against attack by fungal pathogens that enter the plant body by direct penetration through the epidermis. In an approach to enhance resistance of wheat to the powdery mildew fungus Blumeria graminis f.sp. tritici, a novel epidermis-specific promoter was developed and used for expression of two defense-related genes. A 2.3 kb fragment of the wheat GstA1 promoter in combination with an intron-containing part of the wheat WIR1a gene was found to drive strong and constitutive transient expression in wheat epidermis. This promoter-intron combination was used for overexpression of oxalate oxidase 9f-2.8 and TaPERO peroxidase, two defense-related wheat genes expressed in inner leaf tissues. Expression studies of several transgenic lines by in situ oxalate-oxidase staining, RNA and protein blot analyses, as well as real-time PCR, demonstrated strong and constitutive transgene expression in the shoot epidermis. Transient as well as stable over-expression of the TaPERO peroxidase gene in wheat epidermis under the control of the GstA1i promoter resulted in enhanced resistance against Blumeria graminis f.sp. tritici, whereas oxalate-oxidase overexpression had no effect in either system. The data suggest that the wheat GstA1 promoter in combination with the WIR1a intron is useful for transgenic approaches to fungal disease resistance in cereals.     

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.     

A small GTP-binding host protein is required for entry of powdery mildew fungus into epidermal cells of barley

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, sequence-specific 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.     

Protein polyubiquitination plays a role in basal host resistance of barley

To study protein ubiquitination pathways in the interaction of barley (Hordeum vulgare) with the powdery mildew fungus (Blumeria graminis), we measured protein turnover and performed transient-induced gene silencing (TIGS) of ubiquitin and 26S proteasome subunit encoding genes in epidermal cells. Attack by B. graminis hyperdestabilized a novel unstable green fluorescent protein fusion that contains a destabilization domain of a putative barley 1-aminocyclopropane-1-carboxylate synthase, suggesting enhanced protein turnover. Partial depletion of cellular ubiquitin levels by TIGS induced extreme susceptibility of transformed cells toward the appropriate host pathogen B. graminis f. sp hordei, whereas papilla-based resistance to the nonhost pathogen B. graminis f. sp tritici and host resistance mediated by the mlo gene (for mildew resistance locus O) remained unaffected. Cells were rescued from TIGS-induced ubiquitin depletion by synthetic genes encoding wild-type or mutant barley monoubiquitin proteins. The strongest rescue was from a gene encoding a K63R mutant form of ubiquitin blocked in several ubiquitination pathways while still allowing Lys-48-dependent polyubiquitination required for proteasomal protein degradation. Systematic RNA interference of 40 genes encoding all 17 subunits of the proteasome 19S regulatory particle failed to induce hypersusceptibility against B. graminis f. sp hordei. This suggests a role for Lys-48-linked protein polyubiquitination, which is independent from the proteasome pathway, in basal host defense of barley.     

Respiratory burst oxidase homologue A of barley contributes to penetration by the powdery mildew fungus Blumeria graminis f. sp. hordei

Reactive oxygen intermediates (ROI) are closely related to defence reactions of plants against pathogens. A prominent role in the production of ROI has been attributed to the plant respiratory burst oxidase homologues (RBOH) of the human phagocyte GP91(phox). A barley RBOH, which encodes a putative superoxide (O2*-)) producing NADPH oxidase, is described here. Histochemical analysis of the barley-Blumeria graminis f. sp. hordei (Bgh) interaction showed that O(2*-) is produced locally at the site of penetration. In contrast, hydrogen peroxide (H2O2) is produced in non-penetrated cell wall appositions. A barley RBOHA cDNA was isolated and a minor induction of expression of RBOHA was observed during the interactions of barley with Bgh. Transient RNA interference-mediated gene silencing of HvRBOHA during the penetration process of Bgh led to an increase of basal penetration resistance. The results support a potential role of HvRBOHA in cellular accessibility to Blumeria graminis.