Fungal Pathogen Blumeria Graminis Research Articles

Genome-wide association studies on resistance to powdery mildew in cultivated emmer wheat.

Powdery mildew, caused by the fungal pathogen Blumeria graminis (DC.) E. O. Speer f. sp. tritici Em. Marchal (Bgt), is a constant threat to global wheat (Triticum aestivum L.) production. Although ∼100 powdery mildew (Pm) resistance genes and alleles have been identified in wheat and its relatives, more is needed to minimize Bgt's fast evolving virulence. In tetraploid wheat (Triticum turgidum L.), wild emmer wheat [T. turgidum ssp. dicoccoides (Körn. ex Asch. & Graebn.) Thell.] accessions from Israel have contributed many Pm resistance genes. However, the diverse genetic reservoirs of cultivated emmer wheat [T. turgidum ssp. dicoccum (Schrank ex Schübl.) Thell.] have not been fully exploited. In the present study, we evaluated a diverse panel of 174 cultivated emmer accessions for their reaction to Bgt isolate OKS(14)-B-3-1 and found that 66% of accessions, particularly those of Ethiopian (30.5%) and Indian (6.3%) origins, exhibited high resistance. To determine the genetic basis of Bgt resistance in the panel, genome-wide association studies were performed using 46,383 single nucleotide polymorphisms (SNPs) from genotype-by-sequencing and 4331 SNPs from the 9K SNP Infinium array. Twenty-five significant SNP markers were identified to be associated with Bgt resistance, of which 21 SNPs are likely novel loci, whereas four possibly represent emmer derived Pm4a, Pm5a, PmG16, and Pm64. Most novel loci exhibited minor effects, whereas three novel loci on chromosome arms 2AS, 3BS, and 5AL had major effect on the phenotypic variance. This study demonstrates cultivated emmer as a rich source of powdery mildew resistance, and the resistant accessions and novel loci found herein can be utilized in wheat breeding programs to enhance Bgt resistance in wheat.

Resistance to powdery mildew in Irish oat heritage lines

Cereal breeding in Ireland took place in the early twentieth Century to establish native Irish oat cultivars with higher grain yields. However, the production of Irish cultivars declined rapidly from 1973 as the import of international, commercially bred oat cultivars increased which were tested for suitability to Irish climatic conditions. A collection of these heritage oat accessions has been maintained by the Department of Agriculture, Food and the Marine (DAFM). In this study, we investigated a collection of heritage oats for possible resistance to powdery mildew caused by the fungal pathogen Blumeria graminis f. sp. avenae (Bga). We identified Bga resistance in two historical oat accessions (Tyrone 1994 and Sandy). This resistance was mediated by high levels of papillae formation and/or encasements to an Irish single spore isolate. However, intermediate resistance was observed when using a range of Bga isolates.

Control of Blumeria graminis f. sp. hordei on Barley Leaves by Treatment with Fungi-Consuming Protist Isolates

The obligate biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals, which results in large crop losses. Control of B. graminis in barley is mainly achieved by fungicide treatment and by breeding resistant varieties. Vampyrellid amoebae, just like mycophagous protists, are able to consume a variety of fungi. To reveal the impact of some selected fungus-consuming protists on Blumeria graminis f. sp. hordei (Bgh), and to evaluate the possibility of using these protists as biological agents in the future, their feeding behaviour on B. graminis spores on barley leaves was investigated. An experiment was carried out with five different protist isolates (Leptophrys vorax, Platyreta germanica, Theratromyxa weberi U 11, Theratromyxa weberi G7.2 and Acanthamoeba castellanii) and four matched controls, including the food sources of the cultures and the medium. Ten-day-old leaves of barley (Hordeum vulgare cv. Golden Promise) were first inoculated with Blumeria graminis (f. sp. hordei race A6) spores, then treated with protists and fungal colonies on the leaf surfaces were counted under the microscope after 5 days. The isolates L. vorax, P. germanica, and T. weberi U11 did not show a significant reduction in the number of powdery mildew colonies whereas the isolates T. weberi G7.2 and A. castellanii significantly reduced the number of powdery mildew colonies on the leaf surfaces compared to their respective controls. This indicates that these two isolates are capable of reducing B. graminis colonies on barley leaves and are suitable candidates for further investigation for possible use as biological agents. Nevertheless, the susceptibility to dryness and the cell division rate should be considered during the optimisation of the next steps like application procedure and whole plant treatment.

Identification of Quantitative Trait Loci Associated with Powdery Mildew Resistance in Spring Barley under Conditions of Southeastern Kazakhstan.

Barley (Hordeum vulgare L.) is one of the most produced cereal crops in the world. It has traditionally been used for the production of animal feed and for malting, as well as for human consumption. However, its production is highly affected by biotic stress factors, particularly the fungal pathogen Blumeria graminis (DC.) f. sp. hordei (Bgh), which causes powdery mildew (PM). In this study, a collection of 406 barley accessions from the USA, Kazakhstan, Europe, and Africa were assessed for resistance to PM over a 3-year period in southeastern Kazakhstan. The collection was grown in the field in 2020, 2021, and 2022 and was genotyped using the 9K SNP Illumina chip. A genome-wide association study (GWAS) was conducted to identify the quantitative trait loci (QTLs) associated with PM resistance. As a result, seven QTLs for PM resistance were detected on chromosomes 4H, 5H, and 7H (FDR p-values < 0.05). Genetic positions of two QTLs were similar to those of PM resistance QTLs previously reported in the scientific literature, suggesting that the five remaining QTLs are novel putative genetic factors for the studied trait. Haplotype analysis for seven QTLs revealed three haplotypes which were associated with total PM resistance and one haplotype associated with the high PM severity in the barley collection. Identified QTLs and haplotypes associated with the PM resistance of barley may be used for further analysis, trait pyramiding, and marker-assisted selection.

Genetic mapping of adult-plant resistance genes to powdery mildew in triticale

Triticale is a cereal of high economic importance; however, along with the increase in the area of this cereal, it is more often infected by the fungal pathogen Blumeria graminis, which causes powdery mildew. The rapid development of molecular biology techniques, in particular methods based on molecular markers may be an important tool used in modern plant breeding. Development of genetic maps, location of the QTLs defining the region of the genome associated with resistance and selection of markers linked to particular trait can be used to select resistant genotypes as well as to pyramidize several resistance genes in one variety. In this paper, we present a new, high-density genetic map of triticale doubled haploids (DH) population “Grenado” × “Zorro” composed of DArT, silicoDArT, and SNP markers. Composite interval mapping method was used to detect eight QTL regions associated with the area under disease progress curve (AUDPC) and 15 regions with the average value of powdery mildew infection (avPM) based on observation conducted in 3-year period in three different locations across the Poland. Two regions on rye chromosome 4R, and single loci on 5R and 6R were reported for the first time as regions associated with powdery mildew resistance. Among all QTLs, 14 candidate genes were identified coded cyclin-dependent kinase, serine/threonine-protein kinase-like protein as well as AMEIOTIC 1 homolog DYAD-like protein, DETOXIFICATION 16-like protein, and putative disease resistance protein RGA3. Three of identified candidate genes were found among newly described QTL regions associated with powdery mildew resistance in triticale.

The Plant Salicylic Acid Signalling Pathway Regulates the Infection of a Biotrophic Pathogen in Grasses Associated with an Epichloë Endophyte.

The study of the contribution of the plant defence hormones, salicylic acid (SA) and jasmonic acid (JA), in the resistance against pathogens of plants associated with Epichloë fungal endophytes has been scanty. We hypothesised that Epichloë spp., capable of inducing host plant SA-dependent defences, would increase the levels of plant resistance against biotrophic pathogens. Plants of Achnatherum inebrians, with and without the fungal endophyte Epichloë gansuensis, were inoculated with the biotrophic fungal pathogen Blumeria graminis. We measured the status of plant defences (associated with SA and JA signalling pathways) and the levels of resistance to the pathogen. Plants associated with the endophyte showed less disease symptoms caused by the biotrophic pathogen than plants without the endophyte. In agreement with our hypothesis, the Epichloë endophyte increased the plant production of SA and enhanced the expression levels of plant genes of synthesis and response to the SA hormone. The elevated expression of SA-related genes coding for putative plant enzymes with anti-fungal activities promoted by the endophyte may explain the enhanced resistance to the pathogen. The present study highlights that interaction between the plant immune system and Epichloë fungal endophytes can contribute significantly to the resistance of endophyte-symbiotic plants against pathogens.

The leucine-rich repeats in allelic barley MLA immune receptors define specificity towards sequence-unrelated powdery mildew avirulence effectors with a predicted common RNase-like fold.

Nucleotide-binding domain leucine-rich repeat-containing receptors (NLRs) in plants can detect avirulence (AVR) effectors of pathogenic microbes. The Mildew locus a (Mla) NLR gene has been shown to confer resistance against diverse fungal pathogens in cereal crops. In barley, Mla has undergone allelic diversification in the host population and confers isolate-specific immunity against the powdery mildew-causing fungal pathogen Blumeria graminis forma specialis hordei (Bgh). We previously isolated the Bgh effectors AVRA1, AVRA7, AVRA9, AVRA13, and allelic AVRA10/AVRA22, which are recognized by matching MLA1, MLA7, MLA9, MLA13, MLA10 and MLA22, respectively. Here, we extend our knowledge of the Bgh effector repertoire by isolating the AVRA6 effector, which belongs to the family of catalytically inactive RNase-Like Proteins expressed in Haustoria (RALPHs). Using structural prediction, we also identified RNase-like folds in AVRA1, AVRA7, AVRA10/AVRA22, and AVRA13, suggesting that allelic MLA recognition specificities could detect structurally related avirulence effectors. To better understand the mechanism underlying the recognition of effectors by MLAs, we deployed chimeric MLA1 and MLA6, as well as chimeric MLA10 and MLA22 receptors in plant co-expression assays, which showed that the recognition specificity for AVRA1 and AVRA6 as well as allelic AVRA10 and AVRA22 is largely determined by the receptors’ C-terminal leucine-rich repeats (LRRs). The design of avirulence effector hybrids allowed us to identify four specific AVRA10 and five specific AVRA22 aa residues that are necessary to confer MLA10- and MLA22-specific recognition, respectively. This suggests that the MLA LRR mediates isolate-specific recognition of structurally related AVRA effectors. Thus, functional diversification of multi-allelic MLA receptors may be driven by a common structural effector scaffold, which could be facilitated by proliferation of the RALPH effector family in the pathogen genome.

Assessing the Curative and Protective Impacts of Select Fungicides for Control of Powdery Mildew of Wheat.

Powdery mildew, caused by the obligate fungal pathogen Blumeria graminis, has been increasing in incidence and severity on wheat in the Chesapeake Bay region of the mid-Atlantic United States. Although fungicides are used for in-season management of powdery mildew, no studies to date have assessed the ability of these products to control disease after pathogen arrival/infection (curative activity) and the duration of disease control provided after fungicide application (protective activity) under controlled conditions. Five commercially available fungicide products, including Caramba, Stratego YLD, Priaxor, Prosaro, and Trivapro, were applied at either 3 or 5 days after inoculation with B. graminis spores to assess curative activity. In a separate study, protective activity for these fungicides was assessed by applying fungicides and inoculating with B. graminis spores the same day and 21 and 42 days after fungicide application. All fungicides reduced powdery mildew severity on foliage compared with nontreated controls. Priaxor provided 11 to 18% less activity than other fungicides when applied curatively. All products provided protective control up to 42 days after inoculation, with Stratego YLD and Priaxor providing the greatest level of control at 68 and 56%, respectively. Our data indicate that different fungicides can have subtle differences in overall efficacy profiles that may translate to improved control or an extended control window in some situations, but all tested products provide very good to excellent control of powdery mildew on wheat.

Moonlighting Function of Phytochelatin Synthase1 in Extracellular Defense against Fungal Pathogens.

Phytochelatin synthase (PCS) is a key component of heavy metal detoxification in plants. PCS catalyzes both the synthesis of the peptide phytochelatin from glutathione and the degradation of glutathione conjugates via peptidase activity. Here, we describe a role for PCS in disease resistance against plant pathogenic fungi. The pen4 mutant, which is allelic to cadmium insensitive1 (cad1/pcs1) mutants, was recovered from a screen for Arabidopsis mutants with reduced resistance to the nonadapted barley fungal pathogen Blumeria graminis f. sp. hordei PCS1, which is found in the cytoplasm of cells of healthy plants, translocates upon pathogen attack and colocalizes with the PEN2 myrosinase on the surface of immobilized mitochondria. pcs1 and pen2 mutant plants exhibit similar metabolic defects in the accumulation of pathogen-inducible indole glucosinolate-derived compounds, suggesting that PEN2 and PCS1 act in the same metabolic pathway. The function of PCS1 in this pathway is independent of phytochelatin synthesis and deglycination of glutathione conjugates, as catalytic-site mutants of PCS1 are still functional in indole glucosinolate metabolism. In uncovering a peptidase-independent function for PCS1, we reveal this enzyme to be a moonlighting protein important for plant responses to both biotic and abiotic stresses.

The TuMYB46L‐TuACO3 module regulates ethylene biosynthesis in einkorn wheat defense to powdery mildew

Summary Powdery mildew disease, elicited by the obligate fungal pathogen Blumeria graminis f.sp. tritici (Bgt), causes widespread yield losses in global wheat crop. However, the molecular mechanisms governing wheat defense to Bgt are still not well understood.Here we found that TuACO3, encoding the 1‐aminocyclopropane‐1‐carboxylic acid (ACC) oxidase functioning in ethylene (ET) biosynthesis, was induced by Bgt infection of the einkorn wheat Triticum urartu, which was accompanied by increased ET content. Silencing TuACO3 decreased ET production and compromised wheat defense to Bgt, whereas both processes were enhanced in the transgenic wheat overexpressing TuACO3.TuMYB46L, phylogenetically related to Arabidopsis MYB transcription factor AtMYB46, was found to bind to the TuACO3 promoter region in yeast‐one‐hybrid and EMSA experiments. TuMYB46L expression decreased rapidly following Bgt infection. Silencing TuMYB46L promoted ET content and Bgt defense, but the reverse was observed when TuMYB46L was overexpressed.Hence, decreased expression of TuMYB46L permits elevated function of TuACO3 in ET biosynthesis in Bgt‐infected wheat. The TuMYB46L‐TuACO3 module regulates ET biosynthesis to promote einkorn wheat defense against Bgt. Furthermore, we found four chitinase genes acting downstream of the TuMYB46L‐TuACO3 module. Collectively, our data shed a new light on the molecular mechanisms underlying wheat defense to Bgt.

The fungal ribonuclease-like effector protein CSEP0064/BEC1054 represses plant immunity and interferes with degradation of host ribosomal RNA.

The biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals and grasses. We present the first crystal structure of a B. graminis effector of pathogenicity (CSEP0064/BEC1054), demonstrating it has a ribonuclease (RNase)-like fold. This effector is part of a group of RNase-like proteins (termed RALPHs) which comprise the largest set of secreted effector candidates within the B. graminis genomes. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in both monocotyledonous and dicotyledonous plants. CSEP0064/BEC1054 interacts in planta with the pathogenesis-related protein PR10. The effector protein associates with total RNA and weakly with DNA. Methyl jasmonate (MeJA) levels modulate susceptibility to aniline-induced host RNA fragmentation. In planta expression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins (RIPs) that would otherwise lead to host cell death, an unviable interaction and demise of the fungus.

Field grown transgenic Pm3e wheat lines show powdery mildew resistance and no fitness costs associated with high transgene expression.

Pm3 from wheat encodes a nucleotide-binding leucine-rich repeat type of receptor and confers resistance to powdery mildew caused by the fungal pathogen Blumeria graminis f.sp. tritici(Bgt). Each of the 17 functional Pm3 alleles identified so far confers resistance to a distinct spectrum of Bgt isolates. Variant Pm3e has been found in wheat donor line W150 and differs only by two amino acids from the non-functional variant Pm3CS. In order to evaluate the capability of Pm3e to provide powdery mildew field resistance, we generated transgenic Pm3e lines by biolistic transformation of the powdery mildew susceptible spring wheat cultivar Bobwhite. Field trials conducted during four field seasons in Switzerland showed significant and strong powdery mildew resistance of the Pm3e transgenic lines, whereas the corresponding biological sister lines, not containing the transgene, were severely powdery mildew infected. Thus Pm3e alone is responsible for the strong resistance phenotype. The field grown transgenic lines showed high transgene expression and Pm3e protein accumulation with no fitness costs on plant development and yield associated with Pm3e abundance. Line E#1 as well as sister line E#1 showed delayed flowering due to somaclonal variation. The study shows the capability of Pm3e in providing strong powdery mildew field resistance, making its use in wheat breeding programs very promising.

Consistent associations with beneficial bacteria in the seed endosphere of barley (Hordeum vulgare L.)

The importance of the plant microbiome for host fitness has led to the concept of the “plant holobiont”. Seeds are reservoirs and vectors for beneficial microbes, which are very intimate partners of higher plants with the potential to connect plant generations. In this study, the endophytic seed microbiota of numerous barley samples, representing different cultivars, geographical sites and harvest years, was investigated. Cultivation-dependent and -independent analyses, microscopy, functional plate assays, greenhouse assays and functional prediction were used, with the aim of assessing the composition, stability and function of the barley seed endophytic bacterial microbiota. Associations were consistently detected in the seed endosphere with Paenibacillus, Pantoea and Pseudomonas spp., which were able to colonize the root with a notable rhizocompetence after seed germination. In greenhouse assays, enrichment with these bacteria promoted barley growth, improved mineral nutrition and induced resistance against the fungal pathogen Blumeria graminis. We demonstrated here that barley, an important crop plant, was consistently associated with beneficial bacteria inside the seeds. The results have relevant implications for plant microbiome ecology and for the holobiont concept, as well as opening up new possibilities for research and application of seed endophytes as bioinoculants in sustainable agriculture.

Impact of compatible and incompatible barley—Blumeria graminis f.sp. hordei interactions on chlorophyll fluorescence parameters

Interactions between different barley genotypes and the fungal pathogen Blumeria graminis f.sp. hordei (Bgh) have a specific impact on the crop physiology. Within the context of plant resistance phenotyping, it is relevant to investigate early host–pathogen interactions to avoid the crop infestation. Analyzing different parameters of the photosynthesis apparatus gives in-depth information of the plant’s health status and can be used for a spatial and temporal assessment of interaction types during plant–pathogen infestation. In the present study, experiments were performed with a near-isogenic line of barley cv. Ingrid WT (susceptible), mlo3 (papilla-based resistance) as well as a near-isogenic line of cv. Pallas, containing the Mla1 (hypersensitive response-based resistance) gene. After inoculation with Bgh isolate K1, the leaves were measured daily using chlorophyll fluorescence imaging. Inoculated, susceptible wild-type leaves showed a reduced effective quantum yield of the photosystem II (ΦPSII) already 1 day after inoculation. In accordance with the quantum yield reduction, the non-photochemical quenching (NPQ) increased, indicating thermal dissipation of excess energy. The changes of ΦPSII and NPQ represent modifications of the leaf metabolism to aid the fungal nutrition uptake, which is influenced by Bgh. By analyzing these parameters, it was also possible to indicate resistance reactions of mlo3 and Mla1 barley genotypes against Bgh. During papilla formation in mlo3 leaves, ΦPSII revealed the lowest values. In contrast, inoculated Mla1 leaves showed the lowest NPQ. The present study proofs that chlorophyll fluorescence imaging is a valuable tool for investigating early plant–pathogen interaction noninvasively. Furthermore, this phytopathology study uses chlorophyll fluorescence imaging, chlorophyll extraction and microscopic observations to characterize the interaction response of different genotypes to an Bgh infection.

Fine mapping and chromosome walking towards the Ror1 locus in barley (Hordeum vulgare L.)

The Ror1 gene was fine-mapped to the pericentric region of barley chromosome 1HL. Recessively inherited loss-of-function alleles of the barley (Hordeum vulgare) Mildew resistance locus o (Mlo) gene confer durable broad-spectrum disease resistance against the obligate biotrophic fungal powdery mildew pathogen Blumeria graminis f.sp. hordei. Previous genetic analyses revealed two barley genes, Ror1 and Ror2, that are Required for mlo-specified resistance and basal defence. While Ror2 was cloned and shown to encode a t-SNARE protein (syntaxin), the molecular nature or Ror1 remained elusive. Ror1 was previously mapped to the centromeric region of the long arm of barley chromosome 1H. Here, we narrowed the barley Ror1 interval to 0.18cM and initiated a chromosome walk using barley yeast artificial chromosome (YAC) clones, next-generation DNA sequencing and fluorescence in situ hybridization. Two non-overlapping YAC contigs containing Ror1 flanking genes were identified. Despite a high degree of synteny observed between barley and the sequenced genomes of the grasses rice (Oryza sativa), Brachypodium distachyon and Sorghum bicolor across the wider chromosomal area, the genes in the YAC contigs showed extensive interspecific rearrangements in orientation and order. Consequently, the position of a Ror1 homolog in these species could not be precisely predicted, nor was a barley gene co-segregating with Ror1 identified. These factors have prevented the molecular identification of the Ror1 gene for the time being.

The conserved oligomeric Golgi complex is involved in penetration resistance of barley to the barley powdery mildew fungus

Membrane trafficking is vital to plant development and adaptation to the environment. It is suggested that post-Golgi vesicles and multivesicular bodies are essential for plant defence against directly penetrating fungal parasites at the cell wall. However, the actual plant proteins involved in membrane transport for defence are largely unidentified. We applied a candidate gene approach and single cell transient-induced gene silencing for the identification of membrane trafficking proteins of barley involved in the response to the fungal pathogen Blumeria graminis f.sp. hordei. This revealed potential components of vesicle tethering complexes [putative exocyst subunit HvEXO70F-like and subunits of the conserved oligomeric Golgi (COG) complex] and Golgi membrane trafficking (COPIγ coatomer and HvYPT1-like RAB GTPase) as essential for resistance to fungal penetration into the host cell.

A jacalin-related lectin-like gene in wheat is a component of the plant defence system

Jacalin-related lectins (JRLs) are a subgroup of proteins with one or more jacalin-like lectin domains. Although JRLs are often associated with biotic or abiotic stimuli, their biological functions in plants, as well as their relationships to plant disease resistance, are poorly understood. A mannose-specific JRL (mJRL)-like gene (TaJRLL1) that is mainly expressed in stem and spike and encodes a protein with two jacalin-like lectin domains was identified in wheat. Pathogen infection and phytohormone treatments induced its expression; while application of the salicylic acid (SA) biosynthesis inhibitor paclobutrazol and the jasmonic acid (JA) biosynthesis inhibitor diethyldithiocarbamic acid, respectively, substantially inhibited its expression. Attenuating TaJRLL1 through virus-induced gene silencing increased susceptibility to the facultative fungal pathogen Fusarium graminearum and the biotrophic fungal pathogen Blumeria graminis. Arabidopsis thaliana transformed with TaJRLL1 displayed increased resistance to F. graminearum and Botrytis cinerea. JA and SA levels in transgenic Arabidopsis increased significantly. A loss or increase of disease resistance due to an alteration in TaJRLL1 function was correlated with attenuation or enhancement of the SA- and JA-dependent defence signalling pathways. These results suggest that TaJRLL1 could be a component of the SA- and JA-dependent defence signalling pathways.

Regulation of basal resistance by a powdery mildew-induced cysteine-rich receptor-like protein kinase in barley.

The receptor-like protein kinases (RLKs) constitute a large and diverse group of proteins controlling numerous plant physiological processes, including development, hormone perception and stress responses. The cysteine-rich RLKs (CRKs) represent a prominent subfamily of transmembrane-anchored RLKs. We have identified a putative barley (Hordeum vulgare) CRK gene family member, designated HvCRK1. The mature putative protein comprises 645 amino acids, and includes a putative receptor domain containing two characteristic 'domain 26 of unknown function' (duf26) domains in the N-terminal region, followed by a rather short 17-amino-acid transmembrane domain, which includes an AAA motif, two features characteristic of endoplasmic reticulum (ER)-targeted proteins and, finally, a characteristic putative protein kinase domain in the C-terminus. The HvCRK1 transcript was isolated from leaves inoculated with the biotrophic fungal pathogen Blumeria graminis f.sp. hordei (Bgh). HvCRK1 transcripts were observed to accumulate transiently following Bgh inoculation of susceptible barley. Transient silencing of HvCRK1 expression in bombarded epidermal cells led to enhanced resistance to Bgh, but did not affect R-gene-mediated resistance. Silencing of HvCRK1 phenocopied the effective penetration resistance found in mlo-resistant barley plants, and the possible link between HvCRK1 and MLO was substantiated by the fact that HvCRK1 induction on Bgh inoculation was dependent on Mlo. Finally, using both experimental and in silico approaches, we demonstrated that HvCRK1 localizes to the ER of barley cells. The negative effect on basal resistance against Bgh and the functional aspects of MLO- and ER-localized HvCRK1 signalling on Bgh inoculation are discussed.

Barley RIC171 interacts with RACBin plantaand 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.

Transient over‐expression of barley BAX Inhibitor‐1 weakens oxidative defence and MLA12‐mediated resistance to Blumeria graminis f.sp. hordei

SUMMARY BAX Inhibitor-1 (BI-1) is a conserved cell death suppressor protein. In barley, BI-1 (HvBI-1) expression is induced upon powdery mildew infection and when over-expressed in epidermal cells of barley, HvBI-1 induces susceptibility to the biotrophic fungal pathogen Blumeria graminis. We co-expressed mammalian pro-apoptotic BAX together with HvBI-1, and the mammalian BAX antagonist BCL-X(L) in barley epidermal cells. BAX expression led to cessation of cytoplasmic streaming and collapse of the cytoplasm while co-expression of HvBI-1 and BCL-X(L) partially or completely, respectively, rescued cells from BAX lethality. When B. graminis was attacking epidermal cells, a green fluorescent protein fusion of HvBI-1 accumulated at the site of attempted penetration and was also present around haustoria. Over-expression of HvBI-1 in epidermal cells weakened a cell-wall-associated local hydrogen peroxide burst in a resistant mlo-mutant genotype and supported haustoria accommodation in race-specifically resistant MLA12-barley. HvBI-1 is a cell death regulator protein of barley with the potential to suppress host defence reactions.