Barley ROP binding kinase1 is involved in microtubule organization and in basal penetration resistance to the barley powdery mildew fungus

Certain plant receptor-like cytoplasmic kinases were reported to interact with small monomeric G-proteins of the RHO of plant (ROP; also called RAC) family in planta and to be activated by this interaction in vitro. We identified a barley (Hordeum vulgare) partial cDNA of a ROP binding protein kinase (HvRBK1) in yeast (Saccharomyces cerevisiae) two-hybrid screenings with barley HvROP bait proteins. Protein interaction of the constitutively activated (CA) barley HvROPs CA HvRACB and CA HvRAC1 with full-length HvRBK1 was verified in yeast and in planta. Green fluorescent protein-tagged HvRBK1 appears in the cytoplasm and nucleoplasm, but CA HvRACB or CA HvRAC1 can recruit green fluorescent protein-HvRBK1 to the cell periphery. Barley HvRBK1 is an active kinase in vitro, and activity is enhanced by CA HvRACB or GTP-loaded HvRAC1. Hence, HvRBK1 might act downstream of active HvROPs. Transient-induced gene silencing of barley HvRBK1 supported penetration by the parasitic fungus Blumeria graminis f. sp. hordei, suggesting a function of the protein in basal disease resistance. Transient knockdown of HvRBK1 also influenced the stability of cortical microtubules in barley epidermal cells. Hence, HvRBK1 might function in basal resistance to powdery mildew by influencing microtubule organization.     

Genes involved in resistance to powdery mildew in barley differentially modulate root colonization by the mycorrhizal fungus Glomus mosseae

 Arbuscular mycorrhizal fungi (AMF) and Erysiphe graminis are obligate biotrophic fungi with different outcomes in their interaction with plants, different targeted host tissues, but similar patterns of development and infection processes. These similarities raise the question of whether the two types of biotrophic fungal infections have common features in their regulation. To investigate this question, we compared a number of Ror and Rar barley mutants susceptible to E.graminis f. sp. hordei, as well as their resistant progenitors, for susceptibility to infection by the AMF Glomus mosseae. The two powdery mildew-resistant lines BC Ingrid and Sultan presented a similar reduction in G. mosseae development within roots when compared to the wildtype cultivar Ingrid, indicating a systemic effect of the altered genes in the plant. Ror and Rar mutants, in which susceptibility to powdery mildew is restored, showed increased resistance to AM fungal development in their roots when compared to their progenitors, which suggests that corresponding mutations must have affected genes which differentially modulate symbiotic and pathogenic biotrophic plant-fungus interactions. Accepted: 16 September 1999     

Non-host resistance of barley is associated with a hydrogen peroxide burst at sites of attempted penetration by wheat powdery mildew fungus

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 H₂O₂. To elucidate the mechanism of non-host resistance, the accumulation of H₂O₂ in response to Bgt was studied in situ by histochemical staining with diaminobenzidine. H₂O₂ 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.     

The tomato Arp2/3 complex is required for resistance to the powdery mildew fungus Oidium neolycopersici

The actin‐related protein 2/3 complex (Arp2/3 complex), a key regulator of actin cytoskeletal dynamics, has been linked to multiple cellular processes, including those associated with response to stress. Herein, the Solanum habrochaites ARPC3 gene, encoding a subunit protein of the Arp2/3 complex, was identified and characterized. ShARPC3 encodes a 174‐amino acid protein possessing a conserved P21‐Arc domain. Silencing of ShARPC3 resulted in enhanced susceptibility to the powdery mildew pathogen Oidium neolycopersici (On‐Lz), demonstrating a role for ShARPC3 in defence signalling. Interestingly, a loss of ShARPC3 coincided with enhanced susceptibility to On‐Lz, a process that we hypothesize is the result of a block in the activity of SA‐mediated defence signalling. Conversely, overexpression of ShARPC3 in Arabidopsis thaliana, followed by inoculation with On‐Lz, showed enhanced resistance, including the rapid induction of hypersensitive cell death and the generation of reactive oxygen. Heterologous expression of ShARPC3 in the arc18 mutant of Saccharomyces cerevisiae (i.e., ?arc18) resulted in complementation of stress‐induced phenotypes, including high‐temperature tolerance. Taken together, these data support a role for ShARPC3 in tomato through positive regulation of plant immunity in response to O. neolycopersici pathogenesis.     

Patterns of post-infectional protein synthesis in barley carrying different genes for resistance to the powdery mildew fungus

Summary Pairs of susceptible and resistant, near-isogenic cultivars ofHordeum vulgare which differ for the Mla, Mlk and Mlp genes for resistance toErysiphe graminis f. sp.hordei were inoculated with race 3 of this pathogen and patterns of protein synthesis associated with primary infection mapped using pulse-labelling with L-[³⁵S]methionine and 2-dimensional electrophoresis. Extraction of proteins with buffer containing detergent revealed the enhanced synthesis of 5 and 8 polypeptides at 25 and 30 h respectively after inoculation of barley carrying the Mla gene (cvMla). The enhanced synthesis of these same polypeptides together with 11 additional polypeptides was observed at 48 h and 72 h after inoculation of barley carrying either the Mlp (cvMlp) or Mlk (cvMlk) genes. The labelling of several major constitutive polypeptides was suppressed in cvMla at 24 h after inoculation; the labelling of six of these polypeptides was also suppressed in both cvMlp and cvMlk but not until 48 and 72 h after inoculation. These results indicate that changes occur in the synthesis of some common polypeptides following infection of cultivars carrying different resistance genes but the timing and extent of these changes varies with the resistance gene in the host.     

Subunit architecture of the conserved oligomeric Golgi complex

The conserved oligomeric Golgi (COG) complex is thought to function in intra-Golgi retrograde trafficking mediated by coat protein I vesicles, a pathway essential for the proper structure and function of the Golgi apparatus. Previous work suggested that COG might act as a tethering factor to mediate the initial attachment between coat protein I vesicles and Golgi membranes. Here, we present extensive in vitro co-translation and immunoprecipitation experiments leading to a new model for the overall architecture of the mammalian COG complex. The eight COG subunits (Cog1-8) are found to form two heterotrimeric subassemblies (Cog2/3/4 and Cog5/6/7) linked by a heterodimer composed of the remaining subunits (Cog1/8). This model is in excellent agreement with in vivo data presented in an accompanying paper (Oka, T., Vasile, E., Penman, M., Novina, C. D., Dykxhoorn, D. M., Ungar, D., Hughson, F. M., and Krieger, M. (2005) J. Biol. Chem. 280, 32736-32745).     

The conserved oligomeric Golgi complex is required for fucosylation of N-glycans in Caenorhabditis elegans

The conserved oligomeric Golgi complex (COG) is a hetero-octomeric peripheral membrane protein required for retrograde vesicular transport and glycoconjugate biosynthesis within the Golgi. Mutations in subunits 1, 4, 5, 6, 7 and 8 are the basis for a rare inheritable human disease termed congenital disorders of glycosylation type-II. Defects to COG complex function result in aberrant glycosylation, protein trafficking and Golgi structure. The cellular function of the COG complex and its role in protein glycosylation are not completely understood. In this study, we report the first detailed structural analysis of N-glycans from a COG complex-deficient organism. We employed sequential ion trap mass spectrometry of permethylated N-glycans to demonstrate that the COG complex is essential for the formation of fucose-rich N-glycans, specifically antennae fucosylated structures in Caenorhabditis elegans. Our results support the supposition that disruption to the COG complex interferes with normal protein glycosylation in the medial and/or trans-Golgi.     

Single-cell transcript profiling of barley attacked by the powdery mildew fungus

In many plant-pathogen interactions, there are several possible outcomes for simultaneous attacks on the same leaf. For instance, an attack by the powdery mildew fungus on one barley leaf epidermal cell may succeed in infection and formation of a functional haustorium, whereas a neighboring cell attacked at the same time may resist fungal penetration. To date, the mixed cellular responses seen even in susceptible host leaves have made it difficult to relate induced changes in gene expression to resistance or susceptibility in bulk leaf samples. By microextraction of cell-specific mRNA and subsequent cDNA array analysis, we have successfully obtained separate gene expression profiles for specific mildew-resistant and -infected barley cells. Thus, for the first time, it is possible to identify genes that are specifically regulated in infected cells and, presumably, involved in fungal establishment. Further, although much is understood about the genetic basis of effective papilla resistance associated with mutant mlo barley, we provide here the first evidence for gene regulation associated with effective papilla-based nonspecific resistance expressed in nominally "susceptible" wild-type barley.     

Field resistance of spring barley cultivars to powdery mildew in Lithuania

During vegetative period 2004–2005 powdery mildew (Erysiphe graminis DC. f. sp. hordei Em. Marchal) field resistance of spring barley cultivars was investigated at the Lithuanian Institute of Agriculture. The spring barley genotypes tested were Lithuania-registered cultivars, cultivars from genetic resources collection, and the new cultivars used for initial breeding. In total, 23 resistance genes were present in the 84 cultivars studied. Among mono-genes only mlo and 1-B-53 showed very high resistance. Slight powdery mildew necroses (up to 3 scores) formed on cultivars possessing these genes. The maximal powdery mildew (PM) severity reached a score of 8.5 and the area under disease progress curve (AUDPC) a value of 1216.8. The cultivars ‘Primus’, ‘Astoria’, ‘Power’, ‘Harrington’ and ‘Scarlett’ were the most resistant among the non mlo cultivars. Severity of PM on ‘Primus’ reached a score of 3.5 (3.0 of PM necrosis) in average, the other cultivars were diseased from 4.5 (3.0) to 5.0 (2.0). The AUDPC values for these cultivars except ‘Scarlett’ were the lowest (85.0–145.3) among the other cultivars. The highest contrast in development of the other leaf diseases was between highly resistant and susceptible to PM cultivar groups. The fast development of PM depressed development of the other diseases 4.7 times.     

Molecular marker analyses of powdery mildew resistance in barley

Powdery mildew, caused byEryisphe graminis f. sp.hordei, is one of the most important diseases of barley (Hordeum vulgare). A number of loci conditioning resistance to this disease have been reported previously. The objective of this study was to use molecular markers to identify chromosomal regions containing genes for powdery mildew resistance and to estimate the resistance effect of each locus. A set of 28 F₁ hybrids and eight parental lines from a barley diallel study was inoculated with each of five isolates ofE. graminis. The parents were surveyed for restriction fragment length polymorphisms (RFLPs) at 84 marker loci that cover about 1100 cM of the barley genome. The RFLP genotypes of the F₁s were deduced from those of the parents. A total of 27 loci, distributed on six of the seven barley chromosomes, detected significant resistance effects to at least one of the five isolates. Almost all the chromosomal regions previously reported to carry genes for powdery mildew resistance were detected, plus the possible existence of 1 additional locus on chromosome 7. The analysis indicated that additive genetic effects are the most important component in conditioning powdery mildew resistance. However, there is also a considerable amount of dominance effects at most loci, and even overdominance is likely to be present at a number of loci. These results suggest that quantitative differences are likely to exist among alleles even at loci which are considered to carry major genes for resistance, and minor effects may be prevalent in cultivars that are not known to carry major genes for resistance.     

The binary interacting network of the conserved oligomeric Golgi tethering complex

Several recent studies have revealed the existence of a conserved oligomeric Golgi (COG) complex consisting of several novel proteins as well as known Golgi proteins that were identified by independent approaches. The mammalian COG complex contains eight subunits: COG1/LdlBp, COG2/LdlCp, COG3/Sec34, COG4/Cod1, COG5/GTC-90/Cod4, COG6/Cod2, COG7, and COG8/Dor1. COG1, COG2, and COG7 seem structurally unique to mammalian cells, whereas the other five subunits are structurally conserved in yeast, which also contains three other unique proteins (COG1/Sec36p/Cod3p, COG2/Sec35p, and COG7/Cod5p). We report here the network of intermolecular interactions of the COG complex, revealed by in vitro translation and co-immunoprecipitation approaches. Our results suggest that COG4 serves as a core component of the complex by interacting directly with COG1, COG2, COG5, and COG7. COG3 is incorporated by its direct interaction with COG1 and COG2, whereas COG6 and COG8 do not interact with any individual subunit. Incorporation of COG6 into the complex depends on the concerted interaction of both COG5 and COG7, whereas optimal incorporation of COG8 depends on the concerted interaction of COG5, COG6, and COG7. Because COG4 (together with COG1, COG2, and COG3) is among the four essential genes of the COG complex in yeast, this molecular network highlights the structural basis for a crucial role of COG4 in the assembly/function of the complex. A model for the assembly of the COG complex is presented.     

Host surface properties affect prepenetration processes in the barley powdery mildew fungus

The initial contact between Blumeria graminis f.sp. hordei and its host barley (Hordeum vulgare) takes place on epicuticular waxes at the surfaces of aerial plant organs. Here, the extent to which chemical composition, crystal structure and hydrophobicity of cuticular waxes affect fungal prepenetration processes was explored. The leaf surface properties of barley eceriferum (cer) wax mutants were characterized in detail. Barley leaves and artificial surfaces were used to investigate the early events of fungal infection. Even after epicuticular waxes had been stripped away, cer mutant leaf surfaces did not affect fungal prepenetration properties. Removal of total leaf cuticular waxes, however, resulted in a 20% reduction in conidial germination and differentiation. Two major components of barley leaf wax, hexacosanol and hexacosanal, differed considerably in their ability to effectively trigger conidial differentiation on glass surfaces. While hexacosanol, attaining a maximum hydrophobicity with contact angles of no more than 80 degrees, proved to be noninductive, hexacosanal significantly stimulated differentiation in c. 50% of B. graminis conidia, but only at contact angles > 80 degrees. These results, together with an observed inductive effect of highly hydrophobic, wax-free artificial surfaces, provide new insights into the interplay of physical and chemical surface cues involved in triggering prepenetration processes in B. graminis.     

Mapping resistance to powdery mildew in barley reveals a large-effect nonhost resistance QTL

Key message

Resistance factors against non-adapted powdery mildews were mapped in barley. Some QTLs seem effective only to non-adapted mildews, while others also play a role in defense against the adapted form.The durability and effectiveness of nonhost resistance suggests promising practical applications for crop breeding, relying upon elucidation of key aspects of this type of resistance. We investigated which genetic factors determine the nonhost status of barley (Hordeum vulgare L.) to powdery mildews (Blumeria graminis). We set out to verify whether genes involved in nonhost resistance have a wide effectiveness spectrum, and whether nonhost resistance genes confer resistance to the barley adapted powdery mildew. Two barley lines, SusBgt_SC and SusBgt_DC, with some susceptibility to the wheat powdery mildew B. graminis f.sp. tritici (Bgt) were crossed with cv Vada to generate two mapping populations. Each population was assessed for level of infection against four B. graminis ff.spp, and QTL mapping analyses were performed. Our results demonstrate polygenic inheritance for nonhost resistance, with some QTLs effective only to non-adapted mildews, while others play a role against adapted and non-adapted forms. Histology analyses of nonhost interaction show that most penetration attempts are stopped in association with papillae, and also suggest independent layers of defence at haustorium establishment and conidiophore formation. Nonhost resistance of barley to powdery mildew relies mostly on non-hypersensitive mechanisms. A large-effect nonhost resistance QTL mapped to a 1.4 cM interval is suitable for map-based cloning.

     

Sources of powdery mildew resistance in barley landraces from morocco

A total of forty eight accessions of barley landraces from Morocco were screened for resistance to powdery mildew. Twenty two (46%) of tested landraces showed resistance reactions and thirty four single plant lines were selected. Eleven of these lines were tested in seedling stage with seventeen and another twenty three lines with twenty three isolates of powdery mildew respectively. The isolates were chosen according to the virulence spectra observed on the ‘Pallas’ isolines differential set. Line 229–2–2 was identified with resistance to all prevalent in Europe powdery mildew virulence genes. Lines 230–1–1, 248–1–3 showed susceptible reaction for only one and lines 221–3–2, 227–1–1, 244–3–4 for only two isolates respectively. Three different resistance alleles (Mlat, Mla6, and MLA14) were postulated to be present in tested lines alone or in combination. In thirty (88%) tested lines it was impossible to determine which specific gene or genes for resistance were present. Most probably these lines possessed alleles not represented in the ‘Pallas’ isolines differential set. The distribution of reaction type indicated that about 71% of all reaction types observed were classified as powdery mildew resistance (scores 0, 1 and 2). Majority (79%) of resistance reaction types observed in tested lines was intermediate resistance reaction type two and twenty three lines (68%) showed this reaction for inoculation with more than 50% isolates used. The use of new effective sources of resistance from Moroccan barley landraces for diversification of resistance genes for powdery mildew in barley cultivars was discussed.     

Structural analysis of conserved oligomeric Golgi complex subunit 2

The conserved oligomeric Golgi (COG) complex is strongly implicated in retrograde vesicular trafficking within the Golgi apparatus. Although its mechanism of action is poorly understood, it has been proposed to function by mediating the initial physical contact between transport vesicles and their membrane targets. An analogous role in tethering vesicles has been suggested for at least six additional large multisubunit complexes, including the exocyst, a complex essential for trafficking to the plasma membrane. Here we report the solution structure of a large portion of yeast Cog2p, one of eight subunits composing the COG complex. The structure reveals a six-helix bundle with few conserved surface features but a general resemblance to recently determined crystal structures of four different exocyst subunits. This finding provides the first structural evidence that COG, like the exocyst and potentially other tethering complexes, is constructed from helical bundles. These structures may represent platforms for interaction with other trafficking proteins including SNAREs (soluble N-ethylmaleimide factor attachment protein receptors) and Rabs.     

Reconstitution of cyanogenesis in barley (Hordeum vulgare L.) and its implications for resistance against the barley powdery mildew fungus

Barley (Hordeum vulgare L.) produces a leucine-derived cyanogenic β-d-glucoside, epiheterodendrin that accumulates specifically in leaf epidermis. Barley leaves are not cyanogenic, i.e. they do not possess the ability to release hydrogen cyanide, because they lack a cyanide releasing β-d-glucosidase. Cyanogenesis was reconstituted in barley leaf epidermal cells through single cell expression of a cDNA encoding dhurrinase-2, a cyanogenic β-d-glucosidase from sorghum. This resulted in a 35–60% reduction in colonization rate by an obligate parasite Blumeria graminis f. sp. hordei, the causal agent of barley powdery mildew. A database search for barley homologues of dhurrinase-2 identified a (1,4)-β-d-glucan exohydrolase isozyme βII that is located in the starchy endosperm of barley grain. The purified barley (1,4)-β-d-glucan exohydrolase isozyme βII was found to hydrolyze the cyanogenic β-d-glucosides, epiheterodendrin and dhurrin. Molecular modelling of its active site based on the crystal structure of linamarase from white clover, demonstrated that the disposition of the catalytic active amino acid residues was structurally conserved. Epiheterodendrin stimulated appressoria and appressorial hook formation of B. graminis in vitro, suggesting that loss of cyanogenesis in barley leaves has enabled the fungus to utilize the presence of epiheterodendrin to facilitate host recognition and to establish infection.     

Recognition specificity and RAR1/SGT1 dependence in barley Mla disease resistance genes to the powdery mildew fungus

A large number of resistance specificities to the powdery mildew fungus Blumeria graminis f. sp. hordei map to the barley Mla locus. This complex locus harbors multiple members of three distantly related gene families that encode proteins that contain an N-terminal coiled-coil (CC) structure, a central nucleotide binding (NB) site, a Leu-rich repeat (LRR) region, and a C-terminal non-LRR (CT) region. We identified Mla12, which encodes a CC-NB-LRR-CT protein that shares 89 and 92% identical residues with the known proteins MLA1 and MLA6. Slow Mla12-triggered resistance was altered dramatically to a rapid response by overexpression of Mla12. A series of reciprocal domains swaps between MLA1 and MLA6 identified in each protein recognition domain for cognate powdery mildew fungus avirulence genes (AvrMla1 and AvrMla6). These domains were within different but overlapping LRR regions and the CT part. Unexpectedly, MLA chimeras that confer AvrMla6 recognition exhibited markedly different dependence on Rar1, a gene required for the function of some but not all Mla resistance specificities. Furthermore, uncoupling of MLA6-specific function from RAR1 also uncoupled the response from SGT1, a protein known to associate physically with RAR1. Our findings suggest that differences in the degree of RAR1 dependence of different MLA immunity responses are determined by intrinsic properties of MLA variants and place RAR1/SGT1 activity downstream of and/or coincident with the action of resistance protein-containing recognition complexes.     

RMo1 confers blast resistance in barley and is located within the complex of resistance genes containing Mla, a powdery mildew resistance gene

Isolates of Magnaporthe oryzae (the causal agent of rice blast disease) can infect a range of grass species, including barley. We report that barley Hordeum vulgare cv. Baronesse and an experimental line, BCD47, show a range of resistance reactions to infection with two rice blast isolates. The complete resistance of Baronesse to the isolate Ken 54-20 is controlled by a single dominant gene, designated RMo1. RMo1 mapped to the same linkage map position on chromosome 1H as the powdery mildew resistance locus Mla and an expressed sequence tag (k04320) that corresponds to the barley gene 711N16.16. A resistance quantitative trait locus (QTL), at which Baronesse contributed the resistance allele, to the isolate Ken 53-33 also mapped at the same position as RMo1. Synteny analysis revealed that a corresponding region on rice chromosome 5 includes the bacterial blight resistance gene xa5. These results indicate that a defined region on the short arm of barley chromosome 1H, including RMo1 and Mla, harbors genes conferring qualitative and quantitative resistance to multiple pathogens. The partial resistance of BCD47 to Ken53-33 is determined by alleles at three QTL, two of which coincide with the linkage map positions of the mildew resistance genes mlo and Mlf.     

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.     

The isoelectric point of proteins influences their translocation to the extrahaustorial matrix of the barley powdery mildew fungus

Many biotrophic fungal plant pathogens develop feeding structures, haustoria, inside living plant cells, which are essential for their success. Extrahaustorial membranes (EHMs) surround haustoria and delimit the extrahaustorial matrices (EHMxs). Little is known about transport mechanisms across EHMs and what properties proteins and nutrients need in order to cross these membranes. To investigate this further, we expressed fluorescent proteins in the cytosol of infected barley leaf epidermal cells after particle bombardment and investigated properties that influenced their localisation in the powdery mildew EHMx. We showed that this translocation is favoured by a neutral isoelectric point (pI) between 6.0 and 8.4. However, for proteins larger than 50 kDa, pI alone does not explain their localisation, hinting towards a more complex interplay between pI, size, and sequence properties. We discuss the possibility that an EHM translocon is involved in protein uptake into the EHMx.