The race-specific elicitor, NIP1, from the barley pathogen, Rhynchosporium secalis, determines avirulence on host plants of the Rrs1 resistance genotype.

NIP1, a small phytotoxic protein secreted by the barley pathogen Rhynchosporium secalis, is a race-specific elicitor of defense responses in barley cultivars carrying the resistance gene, Rrs1. Co-inoculation employing spores from a virulent fungal race together with the NIP1 protein converted the phenotype of the interaction from compatible to incompatible only on Rrs1-containing plants. In addition, transformation of a virulent fungal race with the nip1 gene yielded avirulent transformants. This demonstrated that the protein is the product of a fungal avirulence gene. The fungal genome was found to contain a single copy of the nip1 gene. Sequence analysis of nip1 cDNA and genomic clones revealed that the gene consists of two exons and one intron. The derived amino acid sequence comprised a secretory signal peptide of 22 amino acids and a cysteine-rich mature protein of 60 amino acids. All fungal races that were avirulent on barley cultivars of the Rrs1 resistance genotype carry and express the nip1 gene and secrete an elicitor-active NIP1 polypeptide. In contrast, races lacking this gene were virulent. In addition, single nucleotide exchanges were detected in the coding region of the nip1 alleles in one virulent fungal race and in a race whose interaction with barley is not controlled by the Rrs1 gene. The resulting exchanges of single amino acids render the gene products elicitor-inactive. Thus, the R.secalis-barley interaction provides the first example of a pathosystem conforming to the gene-for-gene hypothesis in which a plant with a particular resistance gene recognizes a pathogen by a virulence factor, i.e. one of its offensive weapons. On the fungal side, in turn, recognition by the host plant is eluded by either deletion of the encoding gene or alteration of the primary structure of the gene product.     

Molecular population genetic analysis differentiates two virulence mechanisms of the fungal avirulence gene NIP1

Deletion or alteration of an avirulence gene are two mechanisms that allow pathogens to escape recognition mediated by the corresponding resistance gene in the host. We studied these two mechanisms for the NIP1 avirulence gene in field populations of the fungal barley pathogen Rhynchosporium secalis. The product of the avirulence gene, NIP1, causes leaf necrosis and elicits a defense response on plants with the Rrs1 resistance gene. A high NIP1 deletion frequency (45%) was found among 614 isolates from different geographic populations on four continents. NIP1 was also sequenced for 196 isolates, to identify DNA polymorphisms and corresponding NIP1 types. Positive diversifying selection was found to act on NIP1. A total of 14 NIP1 types were found, 11 of which had not been described previously. The virulence of the NIP1 types was tested on Rrs1 and rrs1 barley lines. Isolates carrying three of these types were virulent on the Rrs1 cultivar. One type each was found in California, Western Europe, and Jordan. Additionally, a field experiment with one pair of near-isogenic lines was conducted to study the selection pressure imposed by Rrs1 on field populations of R. secalis. Deletion of NIP1 was the only mechanism used to infect the Rrs1 cultivar in the field experiment. In this first comprehensive study on the population genetics of a fungal avirulence gene, virulence to Rrs1 in R. secalis was commonly achieved through deletion of the NIP1 avirulence gene but rarely also through point mutations in NIP1.     

Heterologous expression of the avirulence gene product, NIP1, from the barley pathogen Rhynchosporium secalis.

NIP1, the product of the avirulence gene AvrRrs1 from Rhynchosporium secalis, a fungal pathogen of barley, is a small secreted cysteine-rich protein. This protein is essential for the specific recognition of the fungus by host plants carrying the complementary resistance gene Rrs1. Different heterologous expression systems were tested to produce sufficient quantities of NIP1 to allow its utilization in receptor identification and isolation. In addition, protein amounts higher than those produced in fungal cultures are required to determine its 3D structure and to analyze its interaction with a receptor. The most efficient method, the synthesis of a His-tag fusion protein in Escherichia coli combined with a refolding procedure, yielded up to 3 mg of recombinant NIP1 from a 1-liter bacterial culture. After removal of the His-tag, the recombinant protein showed the same physicochemical characteristics as the native NIP1 and, most importantly, full biological activity.     

Differential defense reactions in leaf tissues of barley in response to infection by Rhynchosporium secalis and to treatment with a fungal avirulence gene product

Expression of defense-associated genes was analyzed in leaf tissues of near-isogenic resistant and susceptible barley cultivars upon infection by Rhynchosporium secalis. The genes encoding pathogenesis-related (PR) proteins PR-1, PR-5, and PR-9 are specifically expressed in the mesophyll of resistant plants, whereas a germin-like protein (OxOLP) is synthesized in the epidermis irrespective of the resistance genotype. Restriction-mediated differential display was employed to identify additional epidermis-specific genes. This resulted in the detection of another PR gene, PR-10, along with a lipoxygenase gene, LoxA, and a gene of unknown function, pI2-4, which are specifically induced in the epidermis of resistant plants. The gene encoding a putative protease inhibitor, SD10, is preferentially but not exclusively expressed in the epidermis. The fungal avirulence gene product NIP1 triggers the induction of the four PR genes only. At least two additional elicitors, therefore, must be postulated, one for the unspecific induction of OxOLP and one for the resistance-specific induction of LoxA, pI2-4, and SD10. PR-10 expression can be assumed to be the consequence of NIP1 perception by epidermis cells. In contrast, gene expression in the mesophyll is likely to be triggered by an as yet unknown signal that appears to originate in the epidermis and that is strongly amplified in the mesophyll.     

Lost in the middle of nowhere: the AvrLm1 avirulence gene of the Dothideomycete Leptosphaeria maculans

Leptosphaeria maculans, a Dothideomycete causing stem canker on oilseed rape (Brassica napus), develops gene-for-gene interactions with its host plants. To date, nine resistance genes (Rlm1-9) have been identified in Brassica spp. The corresponding nine avirulence genes (AvrLm1-9) in L. maculans have been mapped at four independent loci, thereby revealing two clusters of three and four linked avirulence genes. Here, we report the completion of map-based cloning of AvrLm1. AvrLm1 was genetically delineated within a 7.3 centimorgan interval corresponding to a 439 kb BAC contig. AvrLm1 is a single copy gene isolated within a 269 kb non-coding, heterochromatin-like region. The region comprised a number of degenerated, nested copies of four long-terminal repeat (LTR) retrotransposons, including Pholy and three novel Gypsy-like retrotransposons. AvrLm1 restored the avirulent phenotype on Rlm1 cultivars following functional complementation of virulent isolates. AvrLm1 homologues were not detected in other Leptosphaeria species or in known fungal genomes including the closely related species Stagonospora nodorum. The predicted AvrLm1 protein is composed of 205 amino acids, of which only one is a cysteine residue. It contains a peptide signal suggesting extracellular localization. Unlike most other fungal avirulence genes, AvrLm1 is constitutively expressed, with a probable increased level of expression upon plant infection, suggesting the absence of tight regulation of AvrLm1 expression.     

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.     

Differential recognition of highly divergent downy mildew avirulence gene alleles by RPP1 resistance genes from two Arabidopsis lines

The perception of downy mildew avirulence (Arabidopsis thaliana Recognized [ATR]) gene products by matching Arabidopsis thaliana resistance (Recognition of Peronospora parasitica [RPP]) gene products triggers localized cell death (a hypersensitive response) in the host plant, and this inhibits pathogen development. The oomycete pathogen, therefore, is under selection pressure to alter the form of these gene products to prevent detection. That the pathogen maintains these genes indicates that they play a positive role in pathogen survival. Despite significant progress in cloning plant RPP genes and characterizing essential plant components of resistance signaling pathways, little progress has been made in identifying the oomycete molecules that trigger them. Concluding a map-based cloning effort, we have identified an avirulence gene, ATR1NdWsB, that is detected by RPP1 from the Arabidopsis accession Niederzenz in the cytoplasm of host plant cells. We report the cloning of six highly divergent alleles of ATR1NdWsB from eight downy mildew isolates and demonstrate that the ATR1NdWsB alleles are differentially recognized by RPP1 genes from two Arabidopsis accessions (Niederzenz and Wassilewskija). RPP1-Nd recognizes a single allele of ATR1NdWsB; RPP1-WsB also detects this allele plus three additional alleles with divergent sequences. The Emco5 isolate expresses an allele of ATR1NdWsB that is recognized by RPP1-WsB, but the isolate evades detection in planta. Although the Cala2 isolate is recognized by RPP1-WsA, the ATR1NdWsB allele from Cala2 is not, demonstrating that RPP1-WsA detects a novel ATR gene product. Cloning of ATR1NdWsB has highlighted the presence of a highly conserved novel amino acid motif in avirulence proteins from three different oomycetes. The presence of the motif in additional secreted proteins from plant pathogenic oomycetes and its similarity to a host-targeting signal from malaria parasites suggest a conserved role in pathogenicity.     

Molecular communication between host plant and the fungal tomato pathogenCladosporium fulvum

Host genotype specificity in interactions between biotrophic fungal pathogens and plants in most cases complies with the gene-for-gene model. Success or failure of infection is determined by absence or presence of complementary genes, avirulence and resistance genes, in the pathogen and the host plant, respectively. Resistance, expressed by the induction of a hypersensitive response followed by other defence responses in the host, is envisaged to be based on recognition of the pathogen, mediated through direct interaction between products of avirulence genes of the pathogen (the so-called race-specific elicitors) and receptors in the host plant, the putative products of resistance genes. The interaction between the biothrophic fungusCladosporium fulvum and its only host tomato is a model system to study fungus-plant gene-for-gene relationships. Here we report on isolation, characterization and biological function of putative pathogenicity factors ECP1 and ECP2 and the race-specific elicitors AVR4 and AVR9 ofC. fulvum and cloning and regulation of their encoding genes. Disruption ofecp1 andecp2 genes has no clear effect on pathogenicity ofC. fulvum. Disruption of theavr9 gene, which codes for the race-specific 28 amino acid AVR9 elicitor, in wild type avirulent races, leads to virulence on tomato genotypes carrying the complementary resistance geneCf9. The avirulence geneavr4 encodes a 105 amino acid race-specific elicitor. A single basepair change in the avirulence geneavr4 leads to virulence on tomato genotypes carrying theCf4 resistance gene.     

Marker-based cloning of the region containing the UhAvr1 avirulence gene from the basidiomycete barley pathogen Ustilago hordei

Race-cultivar specialization during the interaction of the basidiomycete smut pathogen Ustilago hordei with its barley host was described in the 1940s. Subsequent genetic analyses revealed the presence of dominant avirulence genes in the pathogen that conform to the gene-for-gene theory. This pathosystem therefore presents an opportunity for the molecular genetic characterization of fungal genes controlling avirulence. We performed a cross between U. hordei strains to obtain 54 progeny segregating for three dominant avirulence genes on three differential barley cultivars. Bulked segregant analysis was used to identify RAPD and AFLP markers tightly linked to the avirulence gene UhAvr1. The UhAvr1 gene is located in an area containing repetitive DNA and this region is undetectable in cosmid libraries prepared from the avirulent parental strain. PCR and hybridization probes developed from the linked markers were therefore used to identify cosmid clones from the virulent (Uhavr1) parent. By walking on Uhavr1-linked cosmid clones, a nonrepetitive, nearby probe was found that recognized five overlapping BAC clones spanning 170 kb from the UhAvr1 parent. A contig of the clones in the UhAvr1 region was constructed and selected probes were used for RFLP analysis of the segregating population. This approach genetically defined an approximately 80-kb region that carries the UhAvr1 gene and provided cloned sequences for subsequent genetic analysis. UhAvr1 represents the first avirulence gene cloned from a basidiomycete plant pathogen.     

Disulfide bond formation and folding of plant peroxidases expressed as inclusion body protein in Escherichia coli thioredoxin reductase negative strains

Escherichia coli is widely used for the production of proteins, which are of interest in structure and function studies. The folding yield of inclusion body protein is, however, generally low (a few percent) for proteins such as the plant and fungal peroxidases, which contain four disulfide bonds, two Ca2+ ions, and a heme group. We have studied the expression yield and folding efficiency of (i) a novel Arabidopsis thaliana peroxidase, ATP N; and (ii) barley grain peroxidase, BP 1. The expression yield ranges from 0 to 60 microgram/ml of cell culture depending on the peroxidase gene and the vector/host combination. The choice of E. coli strain in particular affects the yield of active peroxidase obtained in the folding step. Thus, the yield of active ATP N peroxidase can be increased 50-fold by using thioredoxin reductase negative strains, which facilitate the formation of disulfide bonds in inclusion body protein.     

The problem of how fungal and oomycete avirulence proteins enter plant cells

Recent advances in cloning avirulence genes from a rust fungus and three oomycete species have provided the novel insight that these eukaryotic plant pathogens deliver small proteins into the host cell cytoplasm where they are recognized by resistance proteins. Anne Rehmany et al. have recently identified a potential host-targeting signal in oomycete avirulence proteins from Hyaloperonospora parasitica, Phytophthora sojae and Phytophthora infestans that might be involved in transporting proteins into the host cell. This signal is surprisingly similar to the host targeting signal used by the malaria pathogen Plasmodium fulciparum to target virulence proteins to the mammalian host cell.     

Resistance to <Emphasis Type="Italic">Rhynchosporium commune</Emphasis> in a collection of European spring barley germplasm

Key message

Association analyses of resistance to Rhynchosporium commune in a collection of European spring barley germplasm detected 17 significant resistance quantitative trait loci. The most significant association was confirmed as Rrs1.

Abstract

Rhynchosporium commune is a fungal pathogen of barley which causes a highly destructive and economically important disease known as rhynchosporium. Genome-wide association mapping was used to investigate the genetic control of host resistance to R. commune in a collection of predominantly European spring barley accessions. Multi-year disease nursery field trials revealed 8 significant resistance quantitative trait loci (QTL), whilst a separate association mapping analysis using historical data from UK national and recommended list trials identified 9 significant associations. The most significant association identified in both current and historical data sources, collocated with the known position of the major resistance gene Rrs1. Seedling assays with R. commune single-spore isolates expressing the corresponding avirulence protein NIP1 confirmed that this locus is Rrs1. These results highlight the significant and continuing contribution of Rrs1 to host resistance in current elite spring barley germplasm. Varietal height was shown to be negatively correlated with disease severity, and a resistance QTL was identified that co-localised with the semi-dwarfing gene sdw1, previously shown to contribute to disease escape. The remaining QTL represent novel resistances that are present within European spring barley accessions. Associated markers to Rrs1 and other resistance loci, identified in this study, represent a set of tools that can be exploited by breeders for the sustainable deployment of varietal resistance in new cultivars.

     

Putting knowledge of plant disease resistance genes to work.

Plant disease resistance genes trigger defence mechanisms upon recognition of pathogen compatibility factors, which are encoded by avirulence genes. Isolation of the barley powdery mildew resistance gene Mla opens the door to understanding the extensive allelic diversity of this locus. Completion of the Arabidopsis genome sequence enables the analysis of the complete set of R-gene homologues in a flowering plant. A new R gene, RPW8, conferring resistance in Arabidopsis to powdery mildew, reveals a new class of protein associated with pathogen recognition. New prospects for using R-gene polymorphism in agriculture are becoming apparent.     

The pseudomonas AvrPto protein is differentially recognized by tomato and tobacco and is localized to the plant plasma membrane

The avrPto gene of Pseudomonas syringae pv tomato triggers race-specific resistance in tomato plants carrying Pto, a resistance gene encoding a protein kinase. When introduced into P. s. tabaci, avrPto triggers resistance in tobacco W38 plants that carry the corresponding R gene. The AvrPto protein is believed to be secreted into host cells through the bacterial type III secretion pathway, where it activates disease resistance in tomato by interacting with Pto. We report here the identification of two distinct regions in AvrPto that determine the recognition specificity of this protein in tomato and tobacco. Point mutations in the central region disrupted the avirulence activity in tomato but not in tobacco. Conversely, point mutations in the C-terminal region abolished the avirulence in tobacco but not in tomato. We further report that AvrPto was localized to the plasma membrane of plant cells. Disrupting the membrane association by mutating a putative myristoylation motif of AvrPto abolished the avirulence activity in both tomato and tobacco. These findings demonstrate that AvrPto is recognized differently by the R genes in tomato and tobacco and that the recognition of AvrPto probably is associated with the plasma membrane.     

Haustorially expressed secreted proteins from flax rust are highly enriched for avirulence elicitors

Rust fungi, obligate biotrophs that cause disease and yield losses in crops such as cereals and soybean (Glycine max), obtain nutrients from the host through haustoria, which are specialized structures that develop within host cells. Resistance of flax (Linum usitatissimum) to flax rust (Melampsora lini) involves the induction of a hypersensitive cell death response at haustoria formation sites, governed by gene-for-gene recognition between host resistance and pathogen avirulence genes. We identified genes encoding haustorially expressed secreted proteins (HESPs) by screening a flax rust haustorium-specific cDNA library. Among 429 unigenes, 21 HESPs were identified, one corresponding to the AvrL567 gene. Three other HESPs cosegregated with the independent AvrM, AvrP4, and AvrP123 loci. Expression of these genes in flax induced resistance gene-mediated cell death with the appropriate specificity, confirming their avirulence activity. AvrP4 and AvrP123 are Cys-rich proteins, and AvrP123 contains a Kazal Ser protease inhibitor signature, whereas AvrM contains no Cys residues. AvrP4 and AvrM induce cell death when expressed intracellularly, suggesting their translocation into plant cells during infection. However, secreted AvrM and AvrP4 also induce necrotic responses, with secreted AvrP4 more active than intracellular AvrP4, possibly as a result of enhanced formation of endoplasmic reticulum-dependent disulfide bonds. Addition of an endoplasmic reticulum retention signal inhibited AvrM-induced necrosis, suggesting that both AvrM and AvrP4 can reenter the plant cell after secretion in the absence of the pathogen.     

Recognition of bacterial avirulence proteins occurs inside the plant cell: a general phenomenon in resistance to bacterial diseases?

One of the recent exciting developments in the research area of plant-microbe interactions is a breakthrough in understanding part of the initial signalling between avirulent Gram-negative bacteria and resistant plants. For resistance to occur, both interacting organisms need to express matching genes, the plant resistance gene and the bacterial avirulence gene. The biochemical function of bacterial avirulence genes and the nature of the signal molecules recognized by the plant have been a mystery for a long time. Recently, several laboratories have shown that bacterial avirulence proteins function as elicitors that are perceived within the plant cell.     

The origin and colonization history of the barley scald pathogen Rhynchosporium secalis

The origins of pathogens and their past and present migration patterns are often unknown. We used phylogenetic haplotype clustering in conjunction with model-based coalescent approaches to reconstruct the genetic history of the barley leaf pathogen Rhynchosporium secalis using the avirulence gene NIP1 and its flanking regions. Our results falsify the hypothesis that R. secalis emerged in association with its host during the domestication of barley 10,000 to 15,000 years ago in the Fertile Crescent and was introduced into Europe through the migration of Neolithic farmers. Estimates of time since most recent common ancestor (2500-5000 BP) placed the emergence of R. secalis clearly after the domestication of barley. We propose that modern populations of R. secalis originated in northern Europe following a host switch, most probably from a wild grass onto cultivated barley shortly after barley was introduced into northern Europe. R. secalis subsequently spread southwards into already established European barley-growing areas.