Barley Rom1 reveals a potential link between race-specific and nonhost resistance responses to powdery mildew fungi

The Rar1 gene, identified in the context of race-specific powdery mildew resistance mediated by the Hordeum vulgare (barley) resistance (R) gene Mla12, is required for the function of many R-mediated defense responses in mono- and dicotyledonous plant species. Mla resistance is associated with an oxidative burst and a subsequent cell death reaction of attacked cells. Rar1 mutants are impaired in these responses and, to identify genetic elements which negatively regulate the Mla12-triggered response, we have screened mutagenized Mla12 rar1 mutant populations for restoration of the resistance response. Here we describe the restoration of Mla12-specified resistance (rom1) mutant that restores features of disease resistance to a Blumeria graminis f. sp. hordei isolate expressing the avirulence gene AvrMla12 and retains susceptibility to an isolate lacking AvrMla12. Histochemical analyses show that, in rom1 mutant plants, a whole-cell oxidative burst and cell death response in attacked epidermal cells is restored in the incompatible interaction. Defense responses against tested inappropriate powdery mildews, B. graminis f. sp. tritici and Golovinomyces orontii, were diminished in rar1 mutant plants and enhanced in rom1 mutant plants relative to the wild type. These findings indicate antagonistic activities of Rar1 and Rom1 and reveal their contribution to nonhost and race-specific resistance responses.     

The MLA6 coiled-coil, NBS-LRR protein confers AvrMla6-dependent resistance specificity to Blumeria graminis f. sp. hordei in barley and wheat

The barley Mla locus confers multiple resistance specificities to the obligate fungal biotroph, Blumeria (= Erysiphe) graminis f. sp. hordei. Interspersed within the 240 kb Mla complex are three families of resistance gene homologs (RGHs). Probes from the Mla-RGH1 family were used to identify three classes of cDNAs. The first class is predicted to encode a full-length CC-NBS-LRR protein and the other two classes contain alternatively spliced, truncated variants. Utilizing a cosmid that contains a gene corresponding to the full-length candidate cDNA, two single-cell expression assays were used to demonstrate complementation of AvrMla6-dependent, resistance specificity to B. graminis in barley and wheat. The first of these assays was also used to substantiate previous genetic data that the Mla6 allele requires the signaling pathway component, Rar1, for function. Computational analysis of MLA6 and the Rar1-independent, MLA1 protein reveals 91.2% identity and shows that the LRR domain is subject to diversifying selection. Our findings demonstrate that highly related CC-NBS-LRR proteins encoded by alleles of the Mla locus can dictate similar powdery mildew resistance phenotypes yet still require distinct downstream signaling components.     

DNA polymorphism among barley NILs of cv. Pallas, carrying genes for resistance to powdery mildew (Blumeria graminis f. sp. hordei)

Barley powdery mildew, caused by the pathogen Blumeria graminis f. sp. hordei is an important disease of barley (Hordeum vulgare L.). The random amplified polymorphic DNA (RAPD) method was used to detect DNA polymorphism among 7 Pallas near-isogenic lines (NILs) carrying Mla3, Mla12, Mlk, Mlp, Mlat, Mlg and MlLa genes for resistance to B. graminis f. sp. hordei. From among 500 random 10-mer primers tested, 3 were specific for NIL P2 (Mla3), 1 for P10 (Mla12), 6 for P17 (Mlk), 46 for P19 (Mlp), 4 for P20 (Mlat), 6 for P21 (Mlg), and 4 for P23 (MlLa). The results of this study demonstrated that the RAPD technique is a useful tool for detecting DNA polymorphism among Pallas NILs.     

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.     

Barley MLO modulates actin-dependent and actin-independent antifungal defense pathways at the cell periphery

Cell polarization is a crucial process during plant development, as well as in plant-microbe interactions, and is frequently associated with extensive cytoskeletal rearrangements. In interactions of plants with inappropriate fungal pathogens (so-called non-host interactions), the actin cytoskeleton is thought to contribute to the establishment of effective barriers at the cell periphery against fungal ingress. Here, we impeded actin cytoskeleton function in various types of disease resistance using pharmacological inhibitors and genetic interference via ectopic expression of an actin-depolymerizing factor-encoding gene, ADF. We demonstrate that barley (Hordeum vulgare) epidermal cells require actin cytoskeleton function for basal defense to the appropriate powdery mildew pathogen Blumeria graminis f. sp. hordei and for mlo-mediated resistance at the cell wall, but not for several tested race-specific immune responses. Analysis of non-host resistance to two tested inappropriate powdery mildews, Erysiphe pisi and B. graminis f. sp. tritici, revealed the existence of actin-dependent and actin-independent resistance pathways acting at the cell periphery. These pathways act synergistically and appear to be under negative control by the plasma membrane-resident MLO protein.     

A single-amino acid substitution in the sixth leucine-rich repeat of barley MLA6 and MLA13 alleviates dependence on RAR1 for disease resistance signaling

Interactions between barley and the powdery mildew pathogen, Blumeria graminis f. sp. hordei, (Bgh) are determined by unique combinations of host resistance genes, designated Mildew-resistance locus (Ml), and cognate pathogen avirulence genes. These interactions occur both dependent and independent of Rar1 (required for Mla12 resistance) and Sgt1 (Suppressor of G-two allele of skp1), which are differentially required for diverse plant disease-resistance pathways. We have isolated two new functional Mla alleles, Rar1-independent Mla7 and Rar1-dependent Mla10, as well as the Mla paralogs, Mla6-2 and Mla13-2. Utilizing the inherent diversity amongst Mla-encoded proteins, we identified the only two amino acids exclusively conserved in RAR1-dependent MLA6, MLA10, MLA12, and MLA13 that differ at the corresponding position in RAR1-independent MLA1 and MLA7. Two- and three-dimensional modeling places these residues on a predicted surface of the sixth leucine-rich repeat (LRR) domain at positions distinct from those within the beta-sheets hypothesized to determine resistance specificity. Site-directed mutagenesis of these residues indicates that RAR1 independence requires the presence of an aspartate at position 721, as mutation of this residue to a structurally similar, but uncharged, asparagine did not alter RAR1 dependence. These results demonstrate that a single-amino acid substitution in the sixth MLA LRR can alter host signaling but not resistance specificity to B. graminis.     

Antagonistic control of powdery mildew host cell entry by barley calcium-dependent protein kinases (CDPKs)

Calcium-dependent protein kinases (CDPKs) are known to play pivotal roles in intracellular signaling during abiotic and biotic stress responses. To unravel potential functions of CDPKs in the course of barley (Hordeum vulgare)-powdery mildew (Blumeria graminis) interactions, we systematically analyzed the HvCDPK gene family. We found that, according to the existence of respective expressed sequence tags, at least nine paralogs are expressed in the barley leaf epidermis, the sole target tissue of powdery mildew fungi. We exemplarily selected two HvCDPKs with known full-length coding sequence for functional analysis. Transient expression of a putative constitutive active variant of one of these (HvCDPK4) in Nicotiana benthamiana triggered kinase-dependent mesophyll cell death in tobacco leaves. In a barley mlo mutant genotype, a constitutive active variant of the second paralog, HvCDPK3, partially compromised the highly effective resistance to B. graminis f. sp. hordei. A similar break of mlo resistance was seen upon expression of the junction domain of HvCDPK4, supposed to act as a dominant inhibitor of CDPK activity. Expression of a constitutive active HvCDPK3 or HvCDPK4 form also compromised penetration resistance to the inappropriate wheat powdery mildew fungus. Collectively, our data provide evidence for antagonistic roles of individual CDPK paralogs in the control of host cell entry during the early phase of powdery mildew pathogenesis.     

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.     

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.     

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.     

Expression of resistance to Blumeria graminis f.sp. tritici in 'Chinese Spring' wheat addition lines containing chromosomes from Hordeum vulgare and H. chilense

Blumeria graminis f.sp. tritici (syn. Erysiphe graminis f.sp. tritici) causes an important disease of wheat (powdery mildew) to which Hordeum vulgare and H. chilense are resistant. The study of chromosomal addition lines of H. vulgare and H. chilense in wheat showed that they possessed resistance to wheat powdery mildew. This was expressed as a reduction of disease severity but it was not associated with increased macroscopically visible necrosis. The resistance is of broad genetic basis, conferred by gene(s) present on different chromosomes of both H. vulgare and H. chilense. The feasibility of transferring this resistance to wheat is discussed.     

Gramine increase associated with rapid and transient systemic resistance in barley seedlings induced by mechanical and biological stresses.

Systemic acquired resistance (SAR) is one of the intriguing issues for studying the mechanism in signal transduction system in a whole plant. We found that SAR and increase of an antifungal compound were induced rapidly and transiently in barley (Hordeum vulgare L. cv. Goseshikoku) by mechanical and biological stresses. One of the major antifungal compounds was identified as an indole alkaloid, gramine (N,N-dimethyl-3-aminomethylindole), by mass spectrum and NMR analyses. Gramine is well known as a constitutive compound of barley, but it increased significantly in the primary and secondary leaves of barley seedlings within 12 h after pruning or inoculating with the powdery mildew fungi of barley (Blumeria graminis f.sp. hordei) and wheat (B. graminis f.sp. tritici). However, in the leaf detached from unwounded seedlings or in the leaf inoculated with the barley powdery mildew fungus, gramine did not increase at all. In the water droplets contacted with barley leaves, the amount of leaked gramine increased dependently upon the time after the seedling was injured mechanically. We also found a tight correlation between gramine increase and enhancement of resistance to the barley powdery mildew fungus in barley leaves treated with an endogenous elicitor. Furthermore, such a systemic resistance was not observed in a barley cultivar Morex that lacks the biosynthetic pathway of gramine. From these results, we conclude that gramine is the excellent marker in rapid and transient systemic acquired resistance in barley.