Ubiquitination in Immunity

Race nonspecific resistance of barley against the barley powdery mildew fungus (Blumeria Graminis f.sp. Hordei, Speer, Bgh ) is mediated by recessive mlo alleles and is controlled by at least two additional genes ‘required for ml o‐specified disease resistance’ (Ror1 and Ror2 ). The pathogenesis‐related accumulation of hydrogen peroxide (H2O2) was comparatively analysed in a susceptible barley line (Hordeum vulgare L. Cv Ingrid, genotype Mlo Ror1, Ror2 ), a resistant Ingrid backcross line carrying the mutant allele mlo5 (BCIngrid‐mlo5, genotype mlo5 Ror1 Ror2 ), and in the moderately susceptible mutants A44 and A89 (genotypes mlo5 Ror1 ror2 and mlo5 ror1‐2 Ror2, respectively). In situ localization of H2O2 was performed by microscopic detection of 3,3‐diaminobenzidine (DAB) polymerization. In BCIngrid‐mlo5 , penetration resistance against Bgh attack was closely correlated to H2O2 accumulation in cytoplasmic aggregates and cell wall appositions beneath the appressorium. In contrast, H2O2 accumulation was almost completely absent in susceptible Ingrid. Lines with mutations in Ror genes showed less H2O2 accumulation beneath appressoria, but more interaction sites with whole cell H2O2 accumulation and hypersensitive cell death response than resistant BCIngrid‐mlo5 . Thus, mutations in Ror1 or Ror2 genes influence the cellular pattern of H2O2 accumulation in mlo plants attacked by Bgh . The data support the hypothesis that H2O2 accumulation is involved in resistance to fungal penetration.

The pathogenesis-related accumulation of superoxide radical anions (O·− 2) and hydrogen peroxide (H2O2) was comparatively analyzed in a barley line (Hordeum vulgare L. cv Sultan-5) carrying the powdery mildew (Blumeria graminis f.sp. hordei, Speer, Bgh) resistance gene Mla12, and in susceptible mutants defective in Mla12 or in genes “required for Mla12-specified disease resistance” (Rar1 and Rar2). In-situ localization of reactive oxygen intermediates was performed both by microscopic detection of azide-insensitive nitroblue tetrazolium (NBT) reduction or diaminobenzidine (DAB) polymerization, and by an NBT-DAB double-staining procedure. The Mla12-mediated hypersensitive cell death occurred either in attacked epidermal cells or adjacent mesophyll cells of wild-type plants. Whole-cell H2O2 accumulation was detected in dying cells, while O·− 2 emerged in adjacent cells. Importantly, all susceptible mutants lacked these reactions. An oxalate oxidase, which is known to generate H2O2 and has been implicated in barley resistance against the powdery mildew fungus, was not differentially expressed between the wild type and all mutants. The results demonstrate that the Rar1 and Rar2 gene products, which are control elements of R-gene-mediated programmed cell death, also control accumulation of reactive oxygen intermediates but not the pathogenesis-related expression of oxalate oxidase.

We analysed pathogenesis-related expression of genes, that are assumed to be involved in ubiquitous plant defence mechanisms like the oxidative burst, the hypersensitive cell death reaction (HR) and formation of localized cell wall appositions (papillae). We carried out comparative northern blot and RT-PCR studies with near-isogenic barley (Hordeum vulgareL. cv. Pallas) lines (NILs) resistant or susceptible to the powdery mildew fungus race A6 (Blumeria graminis f.sp. hordei, BghA6). The NILs carrying one of the R-genes Mla12, Mlg or the mlo mutant allele mlo5 arrest fungal development by cell wall appositions (mlo5) or a HR (Mla12) or both (Mlg). Expression of an aspartate protease gene, an ascorbate peroxidase gene and a newly identified cysteine protease gene was up-regulated after inoculation with BghA6, whereas the constitutive expression-level of a BAS gene, that encodes an alkyl hydroperoxide reductase, was reduced. Expression of a newly identified barley homologue of a mammalian cell death regulator, Bax inhibitor 1, was enhanced after powdery mildew inoculation. An oxalate oxidase-like protein was stronger expressed in NILS expressing penetration resistance. A so far unknown gene that putatively encodes the large subunit of a superoxide generating NADPH oxidases was constitutively expressed in barley leaves and its expression pattern did not change after inoculation. A newly identified barley Rac1 homologue was expressed constitutively, such as the functionally linked NADPH oxidase gene. Gene expression patterns are discussed with regard to defence mechanisms and signal transduction.

The role of reactive oxygen intermediate (ROI) accumulation in resistance and susceptibility of plants to parasitic fungi is still little understood. We examined the spatial and temporal occurrence of different ROIs in barley after inoculation with the biotrophic fungus Blumeria graminis f.sp. hordei (Bgh, barley powdery mildew fungus). Using histochemical analyses, we collected correlative data indicating that H2O2 and O2•– play different roles in background penetration resistance to Bgh. To study the role of O2•– in detail, we isolated barley cDNAs encoding a NADPH oxidase GP91PHOX homologue and a RACB homologue, which may be involved in NADPH oxidase activation. Interestingly, transient silencing of RACB or GP91PHOX via sequence-specific RNA interference enhanced penetration resistance of barley to Bgh. Together, data reveal rather a negative than a positive role of superoxide generation in background resistance of barley to Bgh.

Nonhost resistance of cereals to inappropriate formae speciales of Blumeria graminis is little understood. However, on the microscopic level, nonhost defense to B. graminis is reminiscent of host defense preventing fungal development by penetration resistance and the hypersensitive cell death response (HR). We analyzed histochemically the accumulation of superoxide anion radicals (O2•¯) and hydrogen peroxide (H2O2) at sites of B. graminis attack in nonhost barley and wheat. Superoxide visualized by subcellular reduction of nitroblue tetrazolium accumulated in association with successful fungal penetration in attacked cells and in cells neighboring HR. In contrast, H2O2 accumulated in cell wall appositions beneath fungal penetration attempts or in the entire epidermal cell during HR. The data provide evidence for different roles and sources of superoxide and H2O2 in the nonhost interaction of cereals with inappropriate formae speciales of B. graminis.

Non‐host resistance of barley to Blumeria graminis f.sp. tritici (Bgt ), an inappropriate forma specialis of the grass powdery mildew fungus, is associated with formation of cell wall appositions (papillae) at sites of attempted fungal penetration and a hypersensitive cell death reaction (HR) of single attacked cells. Penetration resistance and HR are also typical features of race‐non‐specific and race‐specific resistance of barley to the appropriate Blumeria graminis f.sp. hordei (Bgh ), raising the question of whether genotypic differences in the cellular response of barley to Bgt are detectable. First, we analysed fungal penetration frequencies and HR in different barley accessions known to show altered non‐host resistance. In genotypes with limited resistance to inappropriate cereal rust fungi, we concomitantly detected low penetration resistance to Bgt and significant differences of HR rates during attack from Bgt . Second, we tested barley mutants known to show altered host responses to Bgh . The rar1‐mutation that suppresses many types of race‐cultivar‐specific resistances did not influence the non‐host response of the Bgt‐isolate used in this study. However, mutants of Ror1 and Ror2 , two genes required for full race non‐specific penetration resistance of mlo‐barley to barley powdery mildew fungus, exhibited altered defence response to Bgt , including higher frequencies of fungal penetration. On these mutants, growth of the inappropriate fungus was arrested subsequent to penetration by HR. Together, the data show that barley defence response to the wheat powdery mildew fungus is determined by similar factors as race‐specific and race‐non‐specific resistance to appropriate Bgh.

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 O2·− 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 first line of active defense in plants is triggered by invariant microbial epitopes known as pathogen-associated molecular patterns (PAMPs). Perception of PAMPs by receptors activates a plethora of reactions ending in PAMP-triggered immunity (PTI), which contributes to broad-spectrum resistance 1, 2. Here, we report a homologous triplet of U-box type E3 ubiquitin ligases (PUBs), PUB22, PUB23, and PUB24 in Arabidopsis, that act as negative regulators of PTI in response to several distinct PAMPs. Expression of PUB22/PUB23/PUB24 was induced by PAMPs and infection by pathogens. The pub22/pub23/pub24 triple mutant displayed derepression and impaired downregulation of responses triggered by PAMPs. Immune responses including the oxidative burst, the MPK3 activity, and transcriptional activation of marker genes were increased and/or prolonged. Enhanced activation of PTI responses also resulted in increased resistance against bacterial and oomycete pathogens, which was accompanied by increased production of reactive oxygen species and cell death. Our data provide novel insights into the regulation of immunity in plants and links ubiquitination as a mechanism of negative regulation of PTI.

Plant immune responses require the coordination of a myriad of processes that are triggered upon perception of invading pathogens. Ubiquitin, the ubiquitination system (UBS) and the 26S proteasome are key for the regulation of processes such as the oxidative burst, hormone signaling, gene induction, and programmed cell death. E3 ligases, the specificity determinants of ubiquitination, have received by far the most attention. Several single-unit ligases, which are rapidly induced by biotic cues, function as both positive and negative regulators of immune responses, whereas multisubunit ligases are mainly involved in hormone signaling. An increasing body of evidence emphasizes the heavy targeting of the UBS by pathogen virulence effectors, underlining its importance in immunity.

Piriformospora indica is a root-colonizing basidiomycete that confers a wide range of beneficial traits to its host. The fungus shows a biotrophic growth phase in Arabidopsis (Arabidopsis thaliana) roots followed by a cell death-associated colonization phase, a colonization strategy that, to our knowledge, has not yet been reported for this plant. P. indica has evolved an extraordinary capacity for plant root colonization. Its broad host spectrum encompasses gymnosperms and monocotyledonous as well as dicotyledonous angiosperms, which suggests that it has an effective mechanism(s) for bypassing or suppressing host immunity. The results of our work argue that P. indica is confronted with a functional root immune system. Moreover, the fungus does not evade detection but rather suppresses immunity triggered by various microbe-associated molecular patterns. This ability to suppress host immunity is compromised in the jasmonate mutants jasmonate insensitive1-1 and jasmonate resistant1-1. A quintuple-DELLA mutant displaying constitutive gibberellin (GA) responses and the GA biosynthesis mutant ga1-6 (for GA requiring 1) showed higher and lower degrees of colonization, respectively, in the cell death-associated stage, suggesting that P. indica recruits GA signaling to help establish proapoptotic root cell colonization. Our study demonstrates that mutualists, like pathogens, are confronted with an effective innate immune system in roots and that colonization success essentially depends on the evolution of strategies for immunosuppression.