99th Dahlem conference on infection, inflammation and chronic inflammatory disorders: innate immune responses in plants.

Plants rely exclusively upon mechanisms of innate immunity. Current concepts of the plant innate immune system are based largely on two forms of immunity that engage distinct classes of immune receptors. These receptors enable the recognition of non-self structures that are either conserved between members of a microbial class or specific to individual strains of a microbe. One type of receptor comprises membrane-resident pattern recognition receptors (PRRs) that detect widely conserved microbe-associated molecular patterns (MAMPs) on the cell surface. A second type of mainly intracellular immune sensors, designated resistance (R) proteins, recognizes either the structure or function of strain-specific pathogen effectors that are delivered inside host cells. Phytopathogenic microorganisms have evolved a repertoire of effectors, some of which are delivered into plant cells to sabotage MAMP-triggered immune responses. Plants appear to have also evolved receptors that sense cellular injury by the release and perception of endogenous damage-associated molecular patterns (DAMPs). It is possible that the integration of MAMP and DAMP responses is critical to mount robust MAMP-triggered immunity. This signal integration might help to explain why plants are colonized in nature by remarkably diverse and seemingly asymptomatic microbial communities.

Identification of Genes Required for the Function of Non-Race-Specific mlo Resistance to Powdery Mildew in Barley.

Recessive alleles (mlo) of the Mlo locus in barley mediate a broad, non-race-specific resistance reaction to the powdery mildew fungus Erysiphe graminis f sp hordei. A mutational approach was used to identify genes that are required for the function of mlo. Six susceptible M2 individuals were isolated after inoculation with the fungal isolate K1 from chemically mutagenized seed carrying the mlo-5 allele. Susceptibility in each of these individuals is due to monogenic, recessively inherited mutations in loci unlinked to mlo. The mutants identify two unlinked complementation groups, designated Ror1 and Ror2 (required for mlo-specified resistance). Both Ror genes are required for the function of different tested mlo alleles and for mlo function after challenge with different isolates of E. g. f sp hordei. A quantitative cytological time course analysis revealed that the host cell penetration efficiency in the mutants is intermediate compared with mlo-resistant and Mlo-susceptible genotypes. Ror1 and Ror2 mutants could be differentiated from each other by the same criterion. The spontaneous formation of cell wall appositions in mlo plants, a subcellular structure believed to represent part of the mlo defense, is suppressed in mlo/ror genotypes. In contrast, accumulation of major structural components in the appositions is seemingly unaltered. We conclude that there is a regulatory function for the Ror genes in mlo-specified resistance and propose a model in which the Mlo wild-type allele functions as a negative regulator and the Ror genes act as positive regulators of a non-race-specific resistance response.

Interaction Analyses of Genes Required for Resistance Responses to Powdery Mildew in Barley Reveal Distinct Pathways Leading to Leaf Cell Death.

Race-specific resistance in barley to the powdery mildew fungus (Erysiphe graminis f sp hordei) is associated with a cell death reaction (hypersensitive response [HR]). Genetically, it is dependent on dominant resistance genes (Mlx), and in most cases, it is also dependent on Rar1 and Rar2. Non-race-specific resistance to the fungus, which is due to the lack of the Mlo wild-type allele, is dependent on Ror1 and Ror2 and is not associated with an HR in the region of pathogen attack. However, the absence of the Mlo wild-type allele stimulates a spontaneous cell death response in foliar tissue. This response is also controlled by Ror1 and Ror2, as indicated by trypan blue staining patterns. Lack of Mlo enhances transcript accumulation of pathogenesis-related genes upon fungal challenge, and this response is diminished by mutations in Ror genes. Using DNA marker-assisted selection of genotypes, we provide evidence, via gene interaction studies, that Ror1 and Ror2 are not essential components of race-specific resistance and do not compromise hypersensitive cell death. Reciprocal experiments show that neither is Rar1 a component of mlo-controlled resistance nor does it affect spontaneous cell death. We show that mlo- and Ror-dependent resistance is active when challenged with E. g. f sp tritici, a nonhost pathogen of barley. Our observations suggest separate genetic pathways operating in race-specific and non-race-specific resistance; they indicate also a separate genetic control of hypersensitive and spontaneous cell death in foliar tissue.

Nar-1 and Nar-2, Two Loci Required for Mla12-Specified Race-Specific Resistance to Powdery Mildew in Barley.

Previously isolated susceptible host mutants were used in a genetic and functional study of the resistance response of barley specified by resistance gene Mla12 to the fungal pathogen Erysiphe graminis f sp hordei. Mutant M66 represents a defective allele of Mla12, whereas M22, M82, and M100 represent mutations in loci unlinked to Mla12. Intermutant crosses of the latter three show that susceptibility in M82 and M100 is caused by allelic, recessive mutations that define the Nar-1 gene (necessary for Mla12 resistance gene 1), whereas the semidominant mutation in M22 defines a second unlinked locus, Nar-2. We show that both genes are required for resistance specified by Mlal2 in different genetic backgrounds of barley. Nar-1 maps on barley chromosome 2 within an ~6-centimorgan restriction fragment length polymorphism interval: this is 0.5 centimorgans from the anthocyanin pigmentation gene Ant2. Quantitative cytological analysis showed that functional alleles of Mla12, Nar-1, and Nar-2 are required for triggering a cell death reaction of attacked host cells at early stages during infection. Functional alleles of all three genes were also required for high-level transcript accumulation of barley defense-related genes that encode chitinase, peroxidase, and pathogenesis-related protein-1. The data support the hypothesis that host cell death and high-level accumulation of defense-related gene transcripts, which are under common control of Mla12, Nar-1, and Nar-2, are crucial events of race-specific resistance to powdery mildew.

Defence signalling pathways in cereals.

The combination of mutational and molecular studies has shed light on the role of reactive oxygen intermediates and programmed cell death in cereal disease resistance mechanisms. Rice Rac1 and barley Rar1 represent conserved disease resistance signalling genes, which may have related functions in animals. The analysis of non-pathogenic Magnaporthe grisea mutants may provide novel tools to study host defence pathways.

Closing the ranks to attack by powdery mildew.

Powdery mildews are among the most common plant diseases, infecting over 650 monocot and over 9000 dicot species. Analysis in domesticated barley and wild Arabidopsis has begun to unravel the genetic and molecular frameworks underlying the mechanisms of susceptibility and resistance to these biotrophic fungal pathogens. This has revealed multiple pathways regulating host defense, some of which are also involved in determining the host range of the pathogen. Host-cell death and rapid cell-wall remodeling have emerged as frequent themes in powdery-mildew resistance. Several mutants have been isolated that might shed light on the enigma of susceptibility determinants in plants.

The Mla (powdery mildew) resistance cluster is associated with three NBS-LRR gene families and suppressed recombination within a 240-kb DNA interval on chromosome 5S (1HS) of barley.

Powdery mildew of barley, caused by Erysiphe graminis f. sp. hordei, is a model system for investigating the mechanism of gene-for-gene interaction between large-genome cereals and obligate-fungal pathogens. A large number of loci that confer resistance to this disease are located on the short arm of chromosome 5(1H). The Mla resistance-gene cluster is positioned near the telomeric end of this chromosome arm. AFLP-, RAPD-, and RFLP-derived markers were used to saturate the Mla region in a high-resolution recombinant population segregating for the (Mla6 + Mla14) and (Mla13 + Ml-Ru3) resistance specificities. These tightly linked genetic markers were used to identify and develop a physical contig of YAC and BAC clones spanning the Mla cluster. Three distinct NBS-LRR resistance-gene homologue (RGH) families were revealed via computational analysis of low-pass and BAC-end sequence data derived from Mla-spanning clones. Genetic and physical mapping delimited the Mla-associated, NBS-LRR gene families to a 240-kb interval. Recombination within the RGH families was at least 10-fold less frequent than between markers directly adjacent to the Mla cluster.

Detection of single-copy sequences with digoxigenin-labeled probes in a complex plant genome after separation on pulsed-field gels.

In recent years, the application of rare cutting restriction enzymes, the separation of resulting DNA fragments on pulsed-field gels and the subsequent Southern blot analysis using radioactively labeled probes have been standard laboratory methods to create long-range physical maps of complex genomes. The disadvantages of this technology are the hazardous handling risks when working with radioactivity and long exposure times. In this paper, we describe the use of nonradioactively labeled probes for single-copy sequence detection in a complex plant genome after pulsed-field electrophoretic separation of DNA fragments in the Mbp range. The approach avoids the use of radioactivity and also reduces the exposure time from one to seven days to approximately 2-3 h.

Correlation between hordatine accumulation, environmental factors and genetic diversity in wild barley (Hordeum spontaneum C. Koch) accessions from the Near East Fertile Crescent.

Wild barley shows a large morphological and phenotypic variation, which is associated with ecogeographical factors and correlates with genotypic differences. Diversity of defense related genes and their expression in wild barley has been recognized and has led to attempts to exploit genes from H. spontaneum in breeding programs. The aim of this study was to determine the variation in the accumulation of hordatines, which are Hordeum-specific preformed secondary metabolites with strong and broad antimicrobial activity in vitro, in 50 accessions of H. spontaneum from different habitats in Israel. Differences in the accumulation of hordatines in the seedling stage were significant between different H. spontaneum genotypes from different regional locations and micro-sites. Variation in the hordatine accumulation within genotypes was between 9% and 45%, between genotypes from the same location between 13% and 38%, and between genotypes from different locations up to 121%. Principal component analysis showed that water related factors explain 39%, temperature related factors explain 33% and edaphic factors account for 11% of the observed variation between the populations of H. spontaneum. Genetic analysis of the tested accessions with LP-PCR primers that are specific for genes involved in the biosynthetic pathway of hordatines showed tight correlations between hordatine abundance and genetic diversity of these markers. Multiple regression analyses indicated associations between genetic diversity of genes directly involved in hordatine biosynthesis, ecogeographical factors and the accumulation of hordatines.

Regulators of cell death in disease resistance.

Cell death and disease resistance are intimately connected in plants. Plant disease resistance genes (R genes) are key components in pathogen perception and have a potential to activate cell death pathways. Analysis of R proteins suggests common molecular mechanisms for pathogen recognition and signal emission whereas the subsequent signalling unexpectedly involves a network of pathways of parallel, branching and converging action. Disease resistance signalling mutants have revealed novel types of regulatory proteins whose biochemical functions are still unknown. Accumulation of small molecules such as salicylic acid, reactive oxygen intermediates, and nitric oxide amplifies resistance responses and directs cells to initiate cell death programs. Genetic analyses of lesion mimic mutants provide a glimpse of how cell death thresholds are set via an interplay of positive and negative regulatory components.

A contiguous 60 kb genomic stretch from barley reveals molecular evidence for gene islands in a monocot genome.

The contiguous DNA sequence of a 60 kb genomic interval of barley chromosome 4HL has been assembled. The region harbours a single and novel gypsy -like retrotransposon, designated BAGY-1. Only three genes appear to reside in the genomic stretch. One predicts a plant homologue of ribophorin I, a subunit of the oligosaccharyltransferase-protein complex located in the rough endoplasmatic reticulum. The second is similar to the Drosophila g1 gene encoding a ring finger protein involved in developmental processes. The observed gene density is approximately 5-fold lower than in the best characterized dicot genome of Arabidopsis but 6- to 10-fold higher than expected from an equidistant gene distribution in the complex barley genome. Our data suggest that the 60 kb genomic interval represents part of a gene island, a seemingly distinctive feature of grass genomes.

Structural analyses and dynamics of soluble and cell wall-bound phenolics in a broad spectrum resistance to the powdery mildew fungus in barley.

High pressure liquid chromatography profiles of barley leaf epidermal soluble and cell wall-bound phenolics were analyzed in response to challenge with the fungal pathogen Erysiphe graminis f. sp. hordei. Only one soluble phenolic was found to accumulate differentially in a broad spectrum resistance reaction controlled by mlo resistance alleles in comparison to susceptible near isogenic Mlo lines. Structural analysis identified this compound as a novel phenolic conjugate, p-coumaroyl-hydroxyagmatine (p-CHA). p-CHA but not the nonhydroxylated derivative p-coumaroylagmatine exhibited antifungal activity both in vitro and in vivo. The accumulation of p-CHA in epidermal tissue correlated tightly with fungal penetration attempts of attacked host cells. Furthermore, upon penetration, epidermal cell wall-bound phenolics became resistant to saponification at sites of attempted fungal ingress (papilla), indicating a change in, or the addition of, different chemical bonding types. The switch in saponification sensitivity occurred at least 2 h earlier in the mlo-incompatible than in the Mlo-compatible interaction. Our results suggest that p-CHA and the speed of papillae compaction play important roles in non-race-specific powdery mildew defense.

Chromosome landing at the barley Rar1 locus.

The barley Rar1 gene is an essential component of the race-specific, Mla-12-specified powdery mildew resistance reaction. As part of a map-based cloning strategy designed to isolate Rar1, five barley yeast artificial chromosomes (YACs) have been identified, ranging in size from 300 to 1100 kb. PCR-based YAC end-specific markers have been established and were employed to construct a local YAC contig. Four out of five YAC clones were found to be non-colinear with the source DNA. High-resolution genetic mapping of the YAC ends demonstrated that the set of five overlapping YAC clones encompasses the barley Rar1 gene.

Structural comparison, modes of expression, and putative cis-acting elements of the two 4-coumarate: CoA ligase genes in potato.

4-Coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism in plants, is encoded in potato (Solanum tuberosum L.) by two structurally similar genes (St4cl-1, St4cl-2). Computer-based sequence analyses revealed similarities at the amino acid sequence level with other enzymes dependent on ATP for activation of aromatic carboxylic acids, e.g. some bacterial peptide synthetases. All these enzymes have a common seven amino acid sequence motif containing one cysteine residue. Using an assay on the basis of the polymerase chain reaction, we show that the mRNAs from both 4CL genes accumulate to equal levels in suspension-cultured cells and whole plant tissues, independent of various kinds of activating stimulus applied and of the overall transcriptional activity of the genes. The apparent lack of differential expression, together with the fact that both 4CL genes and proteins are nearly identical in structure, make it unlikely that 4CL isoforms in potato have specific roles in metabolic channeling. Constitutive in vivo footprints in the TATA-box proximal region of the St4cl-1 promoter define putative cis-acting elements which may be involved in the responses of the 4CL genes to various endogenous and exogenous stimuli.

Technical advance. Double-stranded RNA interferes with gene function at the single-cell level in cereals.

Double-stranded RNA (dsRNA) has been shown to specifically interfere with gene function in several organisms including tobacco and the model plant Arabidopsis. Here, we report on rapid and sequence-specific interference of dsRNA with gene function in cereals. Delivery of cognate dsRNA into single epidermal cells of maize, barley or wheat by particle bombardment interfered with the function of co-bombarded UidA (GUS) and TaGLP2a:GFP reporter genes. Cognate dsRNA was also found to specifically interfere with the function of the endogenous genes A1 and Ant18 encoding dihydroflavonol-4-reductase in maize and barley, respectively. Dihydroflavonol-4-reductase is an essential enzyme of the anthocyanin biosynthetic pathway in maize and barley. This pathway can be induced by transient expression of the C1- and b-Peru genes that encode transcription factors. In the presence of dsRNA corresponding to the dihydroflavonol-4-reductase gene, C1- and b-Peru-dependent, cell-autonomous accumulation of red anthocyanin pigments in bombarded cells of maize and barley was reduced. dsRNA was also demonstrated to negatively interfere with Mlo, which encodes a negative regulator of race non-specific resistance to the powdery mildew fungus in barley. In the presence of Mlo dsRNA, transformed cells became more resistant, thereby phenocopying plants that carry a heritable loss-of function mlo resistance allele. The results suggest that direct delivery of dsRNA to cereals leads to a rapid and sequence-specific interference with gene function at the single-cell level.

Molecular cloning, characterization and expression analysis of two catalase isozyme genes in barley.

Clones representing two distinct barley catalase genes, Cat1 and Cat2, were found in a cDNA library prepared from seedling polysomal mRNA. Both clones were sequenced, and their deduced amino acid sequences were found to have high homology with maize and rice catalase genes. Cat1 had a 91% deduced amino acid sequence identity to CAT-1 of maize and 92% to CAT B of rice. Cat2 had 72 and 79% amino acid sequence identities to maize CAT-2 and -3 and 89% to CAT A of rice. Barley, maize or rice isozymes could be divided into two distinct groups by amino acid homologies, with one group homologous to the mitochondria-associated CAT-3 of maize and the other homologous to the maize peroxisomal/glyoxysomal CAT-1. Both barley CATs contained possible peroxisomal targeting signals, but neither had favorable mitochondrial targeting sequences. Cat1 mRNA occurred in whole endosperms (aleurones plus starchy endosperm), in isolated aleurones and in developing seeds, but Cat2 mRNA was virtually absent. Both mRNAs displayed different developmental expression patterns in scutella of germinating seeds. Cat2 mRNA predominated in etiolated seedling shoots and leaf blades. Barley genomic DNA contained two genes for Cat1 and one gene for Cat2. The Cat2 gene was mapped to the long arm of chromosome 4, 2.9 cM in telomeric orientation from the mlo locus conferring resistance to the powdery mildew fungus (Erysiphe graminis f.sp. hordei).

The mlo resistance alleles to powdery mildew infection in barley trigger a developmentally controlled defence mimic phenotype.

Recessive mlo resistance alleles of the Mlo locus in barley control a non race-specific resistance response to infection by the obligate biotrophic fungus Erysiphe graminis f.sp. hordei. All the mlo alleles analysed stop fungal growth at the same developmental stage within a subcellularly restricted, highly localized cell wall apposition directly beneath the site of abortive fungal penetration. We report that near-isogenic lines carrying the alleles mlo1, mlo3 or mlo5 undergo dramatic spontaneous formation of cell wall appositions, not only in the absence of the fungal pathogen but also in sterile grown plants. A comparative study of spontaneous and infection-triggered cell wall appositions reveals a high degree of similarity with respect to structure, chemical composition and distinct localization within plant tissue. We show that the rate of spontaneous apposition formation is dependent on the genetic background of the plant and that its onset is under developmental control. Furthermore, spontaneous formation of wall appositions is specifically triggered by mlo alleles, since it is unaffected in the presence of the race-specific resistance allele Mlg. We propose a model for the function of the Mlo locus that suggests that both Mlo and mlo alleles control qualitatively the same apposition-based resistance mechanism, which, in the presence of the wild-type Mlo allele, is merely less efficient to provide protection against the currently common races of E. graminis f.sp. hordei.

Functional architecture of the light-responsive chalcone synthase promoter from parsley.

We have combined in vivo genomic footprinting and light-induced transient expression of chalcone synthase promoter derivatives in parsley protoplasts to identify cis sequences regulating light activation. The parsley chalcone synthase promoter contains two cis "units" that are light-responsive. Each unit is composed of short DNA stretches of approximately 50 base pairs, and each contains two in vivo footprints. One of the footprints in each unit covers a sequence that is highly conserved among other light- and stress-regulated plant genes. The other footprinted sequences in each unit are not related to each other. The TATA distal light-responsive unit is inherently weak but can compensate partially for the loss of the stronger TATA proximal unit. Levels of light-induced expression from either can be influenced by the presence of a region of approximately 100 base pairs located upstream of the TATA distal light-responsive unit. Combination of the light-responsive units and upstream region generates a synergistic response to light. We speculate that functional compensation generated by nonidentical, but sequence-related, cis units foreshadows combinatorial diversity of cognate trans factors.

Inducible in vivo DNA footprints define sequences necessary for UV light activation of the parsley chalcone synthase gene.

We began characterization of the protein--DNA interactions necessary for UV light induced transcriptional activation of the gene encoding chalcone synthase (CHS), a key plant defense enzyme. Three light dependent in vivo footprints appear on a 90 bp stretch of the CHS promoter with a time course correlated with the onset of CHS transcription. We define a minimal light responsive promoter by functional analysis of truncated CHS promoter fusions with a reporter gene in transient expression experiments in parsley protoplasts. Two of the three footprinted sequence 'boxes' reside within the minimal promoter. Replacement of 10 bp within either of these 'boxes' leads to complete loss of light responsiveness. We conclude that these sequences define the necessary cis elements of the minimal CHS promoter's light responsive element. One of the functionally defined 'boxes' is homologous to an element implicated in regulation of genes involved in photosynthesis. These data represent the first example in a plant defense gene of an induced change in protein--DNA contacts necessary for transcriptional activation. Also, our data argue strongly that divergent light induced biosynthetic pathways share common regulatory units.

Functional borders, genetic fine structure, and distance requirements of cis elements mediating light responsiveness of the parsley chalcone synthase promoter.

The genetic fine structure of cis-acting sequences previously shown to be necessary for light-regulated expression in the promoter of the parsley (Petroselinum crispum) chalcone synthase gene was analyzed. Site-directed mutations and changes in spacing between cis elements were measured in transient expression assays in parsley protoplasts. Clustered point mutations allowed assignment of functional borders. Single-base substitutions within a highly conserved cis element (box II/G box) defined a critical core of seven bases, 5'-ACGTGGC-3'. It is functionally equivalent to a second sequence-related element (box III), which could replace box II in an orientation-dependent manner. The activity of box II required the presence of another juxtaposed element (box I) at a defined distance. No distance requirement was observed between the two large separable promoter regions known to independently confer light-regulated expression. These data support our hypothesis that a cis-acting sequence that is present in a limited number of diversely regulated plant genes gains its functional capacity and specificity by combinatorial diversity involving flanking partner elements.