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.     

Modulation of reactive oxygen species activities and H2O2 accumulation during compatible and incompatible tomato-root-knot nematode interactions

Here, the interaction of Melodoigyne incognita virulent and avirulent pathotypes with susceptible and Mi-resistant tomato (Solanum lycopersicon) has been studied. Significant differences in nematode penetration occurred 2 days postinoculation (dpi) and became stable from 3 dpi onwards. The hypersensitive cell response (HR) in resistant plants prevented the installation of the avirulent pathotype. The virulent pathotype overcame the Mi (nematode) resistance and induced feeding sites in root cells without triggering HR. Reactive oxygen species (ROS), visualized by subcellular reduction of nitroblue tetrazolium, accumulated in nematode penetrated cells. Quantitative analyses with dichlorofluorescein indicated that the oxidative burst occurred very early with both pathotypes, with an enhanced rate in hyper-responsive cells. Hydrogen peroxide (H(2)O(2)), detected by cerium chloride reaction, accumulated in the cell walls and especially in cells neighbouring HR. The apoplastic location of cerium perhydroxide indicated that either the plasma membrane or the cell wall was the primary site of the superoxide/H(2)O(2) generator. The data provide evidence, for the first time, for ROS-generated signals and their spatiotemporal expression in the host and nonhost interaction of tomato with nematodes.     

Functional conservation of wheat and rice Mlo orthologs in defense modulation to the powdery mildew fungus

Homologs of barley Mlo are found in syntenic positions in all three genomes of hexaploid bread wheat, Triticum aestivum, and in rice, Oryza sativa. Candidate wheat orthologs, designated TaMlo-A1, TaMlo-B1, and TaMlo-D1, encode three distinct but highly related proteins that are 88% identical to barley MLO and appear to originate from the three diploid ancestral genomes of wheat. TaMlo-B1 and the rice ortholog, OsMlo2, are able to complement powdery mildew-resistant barley mlo mutants at the single-cell level. Overexpression of TaMlo-B1 or barley Mlo leads to super-susceptibility to the appropriate powdery mildew formae speciales in both wild-type barley and wheat. Surprisingly, overexpression of either Mlo or TaMlo-B1 also mediates enhanced fungal development to tested inappropriate formae speciales. These results underline a regulatory role for MLO and its wheat and rice orthologs in a basal defense mechanism that can interfere with forma specialis resistance to powdery mildews.     

Isozyme Variation and Genetic Distances of Erysiphe graminis DC. Formae Speciales

Abstract Isozymes of ten different enzymes and unspecific stained proteins were used as biochemical genetic markers to study genetic variation within and between E. graminis ff. sp. hordei, avenae, secalis and tritici. In addition, grainproteins of the corresponding host species were examined. In each forma specialis, one genotype proved to be predominant. 131 distinct isozyme and 93 protein bands were distinguishable in these genotypes. However, divergent banding patterns differed only in 8 bands from the predominant banding patterns found within the formae speciales avena, secalis and tritici. The genetic relationships between powdery mildew formae speciales and host species were computed by cluster analysis from similarity (F) and dissimilarity (D) coefficients and illustrated by phylogenetic trees. Marked correspondence was found between E. graminis ff. sp. secalis and tritici (F: 82–90%). Lower homologies were obtained from the comparison ofthese formae speciales respectively with E. graminis ff. sp. hordei (F: 28–34%) and avenae (F: 24–32%). All phylogenetic trees constructed revealed the same arrangement classification of the formae speciales with similar graduation. The comparison of the host species revealed the highest similarity between S. cereale and T. aestivum (F: 74%). Regression analysis confirmed significant correlation between the genetic relationships within host species and powdery mildew formae speciales (r²= 0.81).     

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.     

Characterization of nonhost resistance of Arabidopsis to the Asian soybean rust

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is a devastating disease of soybean. We report the use of the nonhost plant Arabidopsis thaliana to identify the genetic basis of resistance to P. pachyrhizi. Upon attack by P. pachyrhizi, epidermal cells of wild-type Arabidopsis accumulated H2O2, which likely orchestrates the frequently observed epidermal cell death. However, even when epidermal cell death occurred, fungal hyphae grew on and infection was terminated at the mesophyll boundary. These events were associated with expression of PDF1.2, suggesting that P. pachyrhizi, an ostensible biotroph, mimics aspects of a necrotroph. Extensive colonization of the mesophyll occurred in Arabidopsis pen mutants with defective penetration resistance. Although haustoria were found occasionally in mesophyll cells, the successful establishment of biotrophy failed, as evidenced by the cessation of fungal growth. Double mutants affected in either jasmonic acid or salicylic acid signaling in the pen3-1 background revealed the involvement of both pathways in nonhost resistance (NHR) of Arabidopsis to P. pachyrhizi. Interestingly, expression of AtNHL10, a gene that is expressed in tissue undergoing the hypersensitive response, was only triggered in infected pen3-1 mutants. Thus, a suppression of P. pachyrhizi-derived effectors by PEN3 can be inferred. Our results demonstrate that Arabidopsis can be used to study mechanisms of NHR to ASR.     

Multiple avirulence paralogues in cereal powdery mildew fungi may contribute to parasite fitness and defeat of plant resistance

Powdery mildews, obligate biotrophic fungal parasites on a wide range of important crops, can be controlled by plant resistance (R) genes, but these are rapidly overcome by parasite mutants evading recognition. It is unknown how this rapid evolution occurs without apparent loss of parasite fitness. R proteins recognize avirulence (AVR) molecules from parasites in a gene-for-gene manner and trigger defense responses. We identify AVR(a10) and AVR(k1) of barley powdery mildew fungus, Blumeria graminis f sp hordei (Bgh), and show that they induce both cell death and inaccessibility when transiently expressed in Mla10 and Mlk1 barley (Hordeum vulgare) varieties, respectively. In contrast with other reported fungal AVR genes, AVR(a10) and AVR(k1) encode proteins that lack secretion signal peptides and enhance infection success on susceptible host plant cells. AVR(a10) and AVR(k1) belong to a large family with >30 paralogues in the genome of Bgh, and homologous sequences are present in other formae speciales of the fungus infecting other grasses. Our findings imply that the mildew fungus has a repertoire of AVR genes, which may function as effectors and contribute to parasite virulence. Multiple copies of related but distinct AVR effector paralogues might enable populations of Bgh to rapidly overcome host R genes while maintaining virulence.     

Microwounding is a pivotal factor for the induction of actin-dependent penetration resistance against fungal attack

Induced penetration resistance is triggered by failed penetration attempts of nonpathogenic fungi. The resistance mechanism is an important nonhost reaction in plants that can block the invasion of filamentous pathogens such as fungi and oomycetes. However, it remains unclear whether the mechanical stimuli accompanying fungal penetration play a role in induced penetration resistance, whereas the perforation of the cell wall may provide significant stimuli to plant cells. Here, we used microneedles or biolistic bombardment to mimic fungal penetration pegs and a micromanipulation transfer technique of the bio-probe, a germling of Blumeria graminis hordei, to the wounded cells to demonstrate that microwounds derived from fungal penetration attempts may trigger induced penetration resistance in plant cells. When preinoculated with the nonpathogenic fungi Erysiphe pisi and Colletotrichum orbiculare, which were unable to penetrate a barley cell, the penetration of a bio-probe that was transferred by micromanipulation onto the same cell was completely blocked. Fungal penetration was essential to the triggering of induced penetration resistance because a penetration-peg-defective mutant of C. orbiculare completely lacked the ability to trigger resistance. The artificial microwounds significantly, but not completely, blocked the penetration of the bio-probe. Treatment with the actin polymerization inhibitor cytochalasin A or expression of the actin depolymerizing protein HvPro1 caused complete ablation of the induced penetration resistance triggered by either failed fungal penetration or artificial microwounds. These results strongly suggest that microwounding may trigger actin-dependent induced penetration resistance. Manipulation of induced penetration resistance may be a promising target to improve basic disease resistance in plants.     

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.     

Actin Microfilaments are Required for the Expression of Nonhost Resistance in Higher Plants

We investigated the role of actin microfilaments in nonhostresistance of higher plants. Here we present several lines ofevidence to indicate that microfilaments are indeed involvedin blocking fungal penetration of nonhost plants. Erysiphe pisi,a pathogen of pea, normally fails to penetrate into nonhostplants such as barley, wheat, cucumber and tobacco. When tissuesof these nonhost plants were treated with cytochalasins, specificinhibitors of actin polymerization, this fungus became ableto penetrate and formed haustoria in epidermal cells of theseplants. Moreover, treatment of these plants with various kindsand concentrations of cytochalasins allowed several other non-pathogenicfungi, E.graminis hordei, E.graminis tritici, Sphaerotheca fuliginea,Colletotrichum graminicola, My-cosphaella pinodes, C. lagenarium,Altemaria kikuchiana and Corynespora melonis, to also penetratethe cells of these plants. The degree of microfilament depolymeriza-tionvaried depending on the kinds and concentrations of cytochalasinsapplied and we show that this is significantly correlated withthe penetration efficiency of C. graminicola. This indicatesthat the polymerized, filamentous state of actin is necessaryfor plants to block fungal penetration. These results stronglysuggest that actin microfilaments may play important roles inthe expression of nonhost resistance of higher plants. ¹Contribution no. 129 from the Laboratory of Plant Pathology,Mie University     

Nonhost resistance of rice to rust pathogens

Rice is atypical in that it is an agricultural cereal that is immune to fungal rust diseases. This report demonstrates that several cereal rust species (Puccinia graminis f. sp tritici, P. triticina, P. striiformis, and P. hordei) can infect rice and produce all the infection structures necessary for plant colonization, including specialized feeding cells (haustoria). Some rust infection sites are remarkably large and many plant cells are colonized, suggesting that nutrient uptake occurs to support this growth. Rice responds with an active, nonhost resistance (NHR) response that prevents fungal sporulation and that involves callose deposition, production of reactive oxygen species, and, occasionally, cell death. Genetic variation for the efficacy of NHR to wheat stem rust and wheat leaf rust was observed. Unlike cereal rusts, the rust pathogen (Melampsora lini) of the dicotyledenous plant flax (Linum usitatissimum) rarely successfully infects rice due to an apparent inability to recognize host-derived signals. Morphologically abnormal infection structures are produced and appressorial-like structures often don't coincide with stomata. These data suggest that basic compatibility is an important determinate of nonhost infection outcomes of rust diseases on cereals, with cereal rusts being more capable of infecting a cereal nonhost species compared with rust species that are adapted for dicot hosts.     

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.     

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.     

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.     

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

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

A barley ROP GTPase ACTIVATING PROTEIN associates with microtubules and regulates entry of the barley powdery mildew fungus into leaf epidermal cells

Little is known about the function of host factors involved in disease susceptibility. The barley (Hordeum vulgare) ROP (RHO of plants) G-protein RACB is required for full susceptibility of the leaf epidermis to invasion by the biotrophic fungus Blumeria graminis f. sp hordei. Stable transgenic knockdown of RACB reduced the ability of barley to accommodate haustoria of B. graminis in intact epidermal leaf cells and to form hairs on the root epidermis, suggesting that RACB is a common element of root hair outgrowth and ingrowth of haustoria in leaf epidermal cells. We further identified a barley MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN (MAGAP1) interacting with RACB in yeast and in planta. Fluorescent MAGAP1 decorated cortical microtubules and was recruited by activated RACB to the cell periphery. Under fungal attack, MAGAP1-labeled microtubules built a polarized network at sites of successful defense. By contrast, microtubules loosened where the fungus succeeded in penetration. Genetic evidence suggests a function of MAGAP1 in limiting susceptibility to penetration by B. graminis. Additionally, MAGAP1 influenced the polar organization of cortical microtubules. These results add to our understanding of how intact plant cells accommodate fungal infection structures and suggest that RACB and MAGAP1 might be antagonistic players in cytoskeleton organization for fungal entry.     

Cytological and molecular analysis of the Hordeum vulgare-Puccinia triticina nonhost interaction

Cultivated barley, Hordeum vulgare L., is considered to be a nonhost or intermediate host species for the wheat leaf rust fungus Puccinia triticina. Here, we have investigated, at the microscopic and molecular levels, the reaction of barley cultivars to wheat leaf rust infection. In the nonhost resistant cultivar Cebada Capa, abortion of fungal growth occurred at both pre- and posthaustorial stages, suggesting that defense genes are expressed throughout the development of the inappropriate fungus during the nonhost resistance reaction. In the two barley lines L94 and Bowman, a low level of prehaustorial resistance to P. triticina was observed and susceptibility was comparable to that of wheat control plants. Suppression subtractive hybridization was used to identify genes that are differentially expressed during the nonhost resistance reaction in Cebada Capa as well as during the successful establishment of the inappropriate wheat leaf rust fungus in L94. Northern analysis indicated that two candidate genes, including a barley ortholog of the rice resistance gene Xa21, are putatively involved in nonhost and non-race-specific resistance reactions. In addition, a new gene that is specifically induced during the successful development of the inappropriate fungus P. triticina in barley has been identified.     

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.