Comparative Analysis of the Flax Immune Receptors L6 and L7 Suggests an Equilibrium-Based Switch Activation Model.

NOD-like receptors (NLRs) are central components of the plant immune system. L6 is a Toll/interleukin-1 receptor (TIR) domain-containing NLR from flax (Linum usitatissimum) conferring immunity to the flax rust fungus. Comparison of L6 to the weaker allele L7 identified two polymorphic regions in the TIR and the nucleotide binding (NB) domains that regulate both effector ligand-dependent and -independent cell death signaling as well as nucleotide binding to the receptor. This suggests that a negative functional interaction between the TIR and NB domains holds L7 in an inactive/ADP-bound state more tightly than L6, hence decreasing its capacity to adopt the active/ATP-bound state and explaining its weaker activity in planta. L6 and L7 variants with a more stable ADP-bound state failed to bind to AvrL567 in yeast two-hybrid assays, while binding was detected to the signaling active variants. This contrasts with current models predicting that effectors bind to inactive receptors to trigger activation. Based on the correlation between nucleotide binding, effector interaction, and immune signaling properties of L6/L7 variants, we propose that NLRs exist in an equilibrium between ON and OFF states and that effector binding to the ON state stabilizes this conformation, thereby shifting the equilibrium toward the active form of the receptor to trigger defense signaling.

Genome analysis and avirulence gene cloning using a high-density RADseq linkage map of the flax rust fungus, Melampsora lini.

BACKGROUND: Rust fungi are an important group of plant pathogens that cause devastating losses in agricultural, silvicultural and natural ecosystems. Plants can be protected from rust disease by resistance genes encoding receptors that trigger a highly effective defence response upon recognition of specific pathogen avirulence proteins. Identifying avirulence genes is crucial for understanding how virulence evolves in the field. RESULTS: To facilitate avirulence gene cloning in the flax rust fungus, Melampsora lini, we constructed a high-density genetic linkage map using single nucleotide polymorphisms detected in restriction site-associated DNA sequencing (RADseq) data. The map comprises 13,412 RADseq markers in 27 linkage groups that together span 5860 cM and contain 2756 recombination bins. The marker sequences were used to anchor 68.9 % of the M. lini genome assembly onto the genetic map. The map and anchored assembly were then used to: 1) show that M. lini has a high overall meiotic recombination rate, but recombination distribution is uneven and large coldspots exist; 2) show that substantial genome rearrangements have occurred in spontaneous loss-of-avirulence mutants; and 3) identify the AvrL2 and AvrM14 avirulence genes by map-based cloning. AvrM14 is a dual-specificity avirulence gene that encodes a predicted nudix hydrolase. AvrL2 is located in the region of the M. lini genome with the lowest recombination rate and encodes a small, highly-charged proline-rich protein. CONCLUSIONS: The M. lini high-density linkage map has greatly advanced our understanding of virulence mechanisms in this pathogen by providing novel insights into genome variability and enabling identification of two new avirulence genes.

Internalization of flax rust avirulence proteins into flax and tobacco cells can occur in the absence of the pathogen.

Translocation of pathogen effector proteins into the host cell cytoplasm is a key determinant for the pathogenicity of many bacterial and oomycete plant pathogens. A number of secreted fungal avirulence (Avr) proteins are also inferred to be delivered into host cells, based on their intracellular recognition by host resistance proteins, including those of flax rust (Melampsora lini). Here, we show by immunolocalization that the flax rust AvrM protein is secreted from haustoria during infection and accumulates in the haustorial wall. Five days after inoculation, the AvrM protein was also detected within the cytoplasm of a proportion of plant cells containing haustoria, confirming its delivery into host cells during infection. Transient expression of secreted AvrL567 and AvrM proteins fused to cerulean fluorescent protein in tobacco (Nicotiana tabacum) and flax cells resulted in intracellular accumulation of the fusion proteins. The rust Avr protein signal peptides were functional in plants and efficiently directed fused cerulean into the secretory pathway. Thus, these secreted effectors are internalized into the plant cell cytosol in the absence of the pathogen, suggesting that they do not require a pathogen-encoded transport mechanism. Uptake of these proteins is dependent on signals in their N-terminal regions, but the primary sequence features of these uptake regions are not conserved between different rust effectors.

Transformation of the flax rust fungus, Melampsora lini: selection via silencing of an avirulence gene.

Rust fungi cause devastating diseases on many important food crops, with a damaging stem rust epidemic currently affecting wheat production in Africa and the Middle East. These parasitic fungi propagate exclusively on plants, precluding the use of many biotechnological tools available for other culturable fungi. In particular the lack of a stable transformation system has been an impediment to the genetic manipulation required for molecular analysis of rust pathogenicity. We have developed an Agrobacterium-mediated genetic transformation procedure for the model flax rust fungus Melampsora lini, which infects flax (Linum usitatissimum). Selection of transgenic rust lines is based on silencing of AvrL567, which encodes a rust effector protein that is recognised by the flax L6 immune receptor. The non-transgenic rust line is unable to infect flax plants expressing L6, while silenced transgenic lines are virulent on these plants, providing an effective selection system. This directly confirms that the cloned AvrL567 gene is responsible for flax rust virulence phenotypes, and demonstrates the utility of this system to probe rust gene function.

A survey of ß-glucan and arabinoxylan content in wheat.

BACKGROUND: Dietary fibre lowers the risk of coronary heart disease and colorectal cancer. This survey quantifies mixed link ß-glucan (MBG) and arabinoxylan (AX) in wheat and investigates relationships between the grain carbohydrates. MBG and AX contents were measured in 500 and 200 wheat accessions respectively, including diploid, tetraploid and hexaploid genotypes comprising primitive, synthetic and elite lines. RESULTS: Overall, MBG contents ranged between 1.8 and 18.0 g kg(-1) grain dry weight. In wheat-barley addition lines and triticale hexaploids the levels were 9.0-11.3 and 3.5-9.6 g kg(-1) respectively. The amounts in synthetic wheats were nearer their tetraploid parents than their diploid parents. AX and total non-starch polysaccharide (NSP) contents ranged from 23.7 to 107.5 g kg(-1) and from 31.7 to 136.7 g kg(-1) respectively. Linear regressions showed that the relationships of starch and grain weight with NSP glucose were stronger than those with AX. CONCLUSION: The results indicated insufficient genetic diversity in the germplasm surveyed to initiate a breeding programme to increase the amount of MBG in wheat grain to 20 g kg(-1) , a level considered high enough to confer a 10-15% reduction in blood cholesterol.

Diversity and evolution of effector loci in natural populations of the plant pathogen Melampsora lini.

Genetic variation for pathogen infectivity is an important driver of disease incidence and prevalence in both natural and managed systems. Here, we use the interaction between the rust pathogen, Melampsora lini, and two host plants, Linum marginale and Linum usitatissimum, to examine how host-pathogen interactions influence the maintenance of polymorphism in genes underlying pathogen virulence. Extensive sequence variation at two effector loci (AvrP123, AvrP4) was found in M. lini isolates collected from across the native range of L. marginale in Australia, as well as in isolates collected from a second host, the cultivated species L. usitatissimum. A highly significant excess of nonsynonymous compared with synonymous polymorphism was found at both loci, suggesting that diversifying selection is important for the maintenance of the observed sequence diversity. Agrobacterium-mediated transient transformation assays were used to demonstrate that variants of both the AvrP123 and AvrP4 genes are differentially recognized by resistance genes in L. marginale. We further characterized patterns of nucleotide variation at AvrP123 and AvrP4 in 10 local populations of M. lini infecting the wild host L. marginale. Populations were significantly differentiated with respect to allelic representation at the Avr loci, suggesting the possibility of local selection maintaining distinct genetic structures between pathogen populations, whereas limited diversity may be explained via selective sweeps and demographic bottlenecks. Together, these results imply that interacting selective and nonselective factors, acting across a broad range of scales, are important for the generation and maintenance of adaptively significant variation in populations of M. lini.

The role of secreted proteins in diseases of plants caused by rust, powdery mildew and smut fungi.

Five unrelated avirulence (Avr) gene families have been cloned from flax rust and barley powdery mildew, fungal pathogens that make close contact with living host plant cells using specialized feeding structures called haustoria. Transgenic expression studies indicate Avr proteins are recognized by disease resistance (R) proteins within host cells, which suggests that Avr proteins are transported via an as yet unidentified route from the fungus to the host during infection. Recognition of flax rust AvrL567 proteins is by direct R-Avr protein interaction. Virulence effector functions have been demonstrated for barley powdery mildew Avr proteins Avra10 and Avrk1. Mildew resistance triggered by Avra10 in barley involves association of the cognate barley R protein Mla10 and transcriptional repressor proteins, including HvWRKY2, in the host nucleus. High amplitude defence gene expression has a dual dependence on transcriptional de-repression induced by specific R-Avr protein recognition and additionally, activation signals initiated by host perception of general pathogen molecules.

Flax rust resistance gene specificity is based on direct resistance-avirulence protein interactions.

Genetic studies of the flax-flax rust interaction led to the formulation of the gene-for-gene hypothesis and identified resistance genes (R) in the host plant and pathogenicity genes, including avirulence (Avr) and inhibitor of avirulence genes (I), in the rust pathogen. R genes have now been cloned from four of the five loci in flax and all encode proteins of the Toll, Interleukin-1 receptor, R gene-nucleotide binding site-leucine-rich repeat (TIR-NBS-LRR) class. Avr genes have been cloned from four loci in flax rust and encode small secreted proteins with no between locus similarity and no close homologs in current data bases. It is postulated that Avr proteins enter the host cell, have virulence effector functions, and in resistant host genotypes, are recognized by direct and specific interaction with host R proteins, leading to activation of rust resistance defense responses. Direct interaction between R and Avr proteins is the basis of gene-for-gene specificity in the flax-flax rust system and both R and Avr genes have the signatures of diversifying selection, suggesting the existence of a coevolutionary arms race between the host plant and its obligate rust pathogen.

Flax rust infection transcriptomics reveals a transcriptional profile that may be indicative for rust Avr genes.

Secreted effectors of fungal pathogens are essential elements for disease development. However, lack of sequence conservation among identified effectors has long been a problem for predicting effector complements in fungi. Here we have explored the expression characteristics of avirulence (Avr) genes and candidate effectors of the flax rust fungus, Melampsora lini. We performed transcriptome sequencing and real-time quantitative PCR (qPCR) on RNA extracted from ungerminated spores, germinated spores, isolated haustoria and flax seedlings inoculated with M. lini isolate CH5 during plant infection. Genes encoding two categories of M. lini proteins, namely Avr proteins and plant cell wall degrading enzymes (CWDEs), were investigated in detail. Analysis of the expression profiles of 623 genes encoding predicted secreted proteins in the M. lini transcriptome shows that the six known Avr genes (i.e. AvrM (avrM), AvrM14, AvrL2, AvrL567, AvrP123 (AvrP) and AvrP4) fall within a group of 64 similarly expressed genes that are induced in planta and show a peak of expression early in infection with a subsequent decline towards sporulation. Other genes within this group include two paralogues of AvrL2, an AvrL567 virulence allele, and a number of genes encoding putative effector proteins. By contrast, M. lini genes encoding CWDEs fall into different expression clusters with their distribution often unrelated to their catalytic activity or substrate targets. These results suggest that synthesis of M. lini Avr proteins may be regulated in a coordinated fashion and that the expression profiling-based analysis has significant predictive power for the identification of candidate Avr genes.

Structural and functional insights into the modulation of the activity of a flax cytokinin oxidase by flax rust effector AvrL567-A.

During infection, plant pathogens secrete effector proteins to facilitate colonization. In comparison with our knowledge of bacterial effectors, the current understanding of how fungal effectors function is limited. In this study, we show that the effector AvrL567-A from the flax rust fungus Melampsora lini interacts with a flax cytosolic cytokinin oxidase, LuCKX1.1, using both yeast two-hybrid and in planta bimolecular fluorescence assays. Purified LuCKX1.1 protein shows catalytic activity against both N6-(Δ2-isopentenyl)-adenine (2iP) and trans-zeatin (tZ) substrates. Incubation of LuCKX1.1 with AvrL567-A results in increased catalytic activity against both substrates. The crystal structure of LuCKX1.1 and docking studies with AvrL567-A indicate that the AvrL567 binding site involves a flexible surface-exposed region that surrounds the cytokinin substrate access site, which may explain its effect in modulating LuCKX1.1 activity. Expression of AvrL567-A in transgenic flax plants gave rise to an epinastic leaf phenotype consistent with hormonal effects, although no difference in overall cytokinin levels was observed. We propose that, during infection, plant pathogens may differentially modify the levels of extracellular and intracellular cytokinins.

Crystal structure of the Melampsora lini effector AvrP reveals insights into a possible nuclear function and recognition by the flax disease resistance protein P.

The effector protein AvrP is secreted by the flax rust fungal pathogen (Melampsora lini) and recognized specifically by the flax (Linum usitatissimum) P disease resistance protein, leading to effector-triggered immunity. To investigate the biological function of this effector and the mechanisms of specific recognition by the P resistance protein, we determined the crystal structure of AvrP. The structure reveals an elongated zinc-finger-like structure with a novel interleaved zinc-binding topology. The residues responsible for zinc binding are conserved in AvrP effector variants and mutations of these motifs result in a loss of P-mediated recognition. The first zinc-coordinating region of the structure displays a positively charged surface and shows some limited similarities to nucleic acid-binding and chromatin-associated proteins. We show that the majority of the AvrP protein accumulates in the plant nucleus when transiently expressed in Nicotiana benthamiana cells, suggesting a nuclear pathogenic function. Polymorphic residues in AvrP and its allelic variants map to the protein surface and could be associated with differences in recognition specificity. Several point mutations of residues on the non-conserved surface patch result in a loss of recognition by P, suggesting that these residues are required for recognition.

The genome sequence and effector complement of the flax rust pathogen Melampsora lini.

Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed most extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp). Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimize parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analyzed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote infection on their hosts.

Relationships between rust resistance genes at the M locus in flax.

Genes at the M locus in flax (Linum usitatissimum) that confer resistance to flax rust (Melampsora lini) occur in complex haplotypes containing up to 15 related genes or gene fragments. We have cloned two additional functional resistance genes at this locus, M1 and M3, by transposon tagging and candidate gene approaches, and investigated the genetic relationships between four genes (M, M1, M3 and M4) by recombination analysis. M1 and M3, like M, are members of the nucleotide binding site, leucine-rich repeat (NBS-LRR) family. Comparisons of the predicted M1 and M3 amino acid sequences with M and L6 reveal that: (i) M1 contains four additional LRRs, probably as a result of an unequal crossover event between duplicated regions; (ii) M1 shares large segments of exact identity with M and M3, indicative of intragenic recombination events; and (iii) a large number of amino acid differences are scattered throughout the M, M1 and M3 proteins. Recombination analysis (here and in previous studies) has revealed that M readily recombines with M1, M3 and M4, whereas these three genes fail to recombine despite large family sizes (>5800) in two test-cross families, suggesting that they may occupy allelic positions in the gene cluster. Several restriction fragment length polymorphism markers within or near the M locus were mapped with respect to seven crossover events between M and M1. The results of this and previous studies provide evidence of structural differences between: (i) homoeologous loci in the different genomes of flax; (ii) different haplotypes at the M locus; (iii) different resistance genes in the M group; and (iv) the flanking regions downstream of M locus resistance genes.

Further analysis of gene-for-gene disease resistance specificity in flax.

SUMMARY The flax rust resistance gene L, a nucleotide binding site, leucine-rich repeat (NBS-LRR) class of plant resistance gene, has 12 characterized alleles with different gene-for-gene resistance specificities. Here the specificities of presumptive L1, L5, L8 and L11 genomic clones are confirmed by transgenic expression. L6 and L11 differ by 33 amino acids, 32 in the LRR region and one in the C-terminal non-LRR region, and recognize unrelated avirulence proteins, AvrL567 and AvrL11, respectively. To analyse the specificity differences, 13 L6L11 recombinant genes were constructed in vitro and tested in transgenic flax for resistance to F2 progeny of rust strain CH5, in which the unlinked avirulence genes AvrL567 and AvrL11 segregate. The data show that the single C-terminal non-LRR region polymorphism is not involved in L6-L11 specificity differences, that polymorphisms necessary for specificity are spread throughout the LRR region and that some polymorphisms essential for L11 are not essential for L6. Seven 'null' recombinants expressed no resistance when tested with CH5-derived rusts. These were tested for new resistance specificities by inoculation with a strain of rust, Bs-1, which is distantly related to CH5 and which potentially carries a different range of avirulence specificities. The 'null' recombinant L6L11RV, which differs from L6 and L11 by its susceptibility to CH5, was resistant to strain Bs-1. The specificity difference is due to a reduction in the number of AvrL567 variants recognized by L6L11RV compared with L6 and not due to recognition of an unrelated Avr gene product in strain Bs-1.

Rust of flax and linseed caused by Melampsora lini.

SUMMARY: Melampsora lini, while of economic importance as the causal agent of rust disease of flax and linseed, has for several decades been the 'model' rust species with respect to genetic studies of avirulence/virulence. Studies by Harold Flor demonstrated that single pairs of allelic genes determine the avirulence/virulence phenotype on host lines with particular resistance genes and led him to propose his famous 'gene-for-gene' hypothesis. Flor's inheritance studies, together with those subsequently carried out by others, also revealed that, in some cases, an inhibitor gene pair and an avirulence/virulence gene pair interact to determine the infection outcome on host lines with particular resistance genes. Recently, avirulence/virulence genes at four loci, AvrL567, AvrM, AvrP4 and AvrP/AvrP123, have been cloned. All encode novel, small, secreted proteins that are recognized inside plant cells. Yeast two-hybrid studies have shown that the AvrL567 proteins interact directly with the resistance gene protein. The molecular basis of Flor's gene-for-gene relationship has now been elucidated for six interacting gene pairs: those involving resistance genes L5, L6, L7, M, P and P2, where both the resistance gene and the corresponding avirulence gene have been cloned. In other inheritance studies it has been shown that M. lini does not possess a (+) and (-) mating system, but may possess a two factor system. Double-stranded (ds) RNA molecules occur in many strains of M. lini: examination of the progeny of one strain that possesses 11 dsRNA molecules revealed that they fall into three transmission units, designated L, A and B. The L unit consists of a single large dsRNA of 5.2 kbp while the A and B units each consist of five dsRNAs in the size range 1.1-2.8 kbp. The three units have different sexual and asexual transmission characteristics. The L unit is encapsidated in a virus-like particle, whereas the other units are not encapsidated. The population and coevolutionary aspects of M. lini on a wild, native Australian host species, Linum marginale, have been extensively investigated. A recent molecular analysis revealed that the M. lini isolates from L. marginale fall into two distinct lineages, one of which is apparently hybrid between two diverse genomes. Isolates in this lineage are largely fixed for heterozygosity, which suggests that sexual recombination does not occur in this lineage.

Crystal structures of flax rust avirulence proteins AvrL567-A and -D reveal details of the structural basis for flax disease resistance specificity.

The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4- and 2.3-A resolution, respectively. The structures of both proteins are very similar and reveal a beta-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.

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.

Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes.

Plant resistance proteins (R proteins) recognize corresponding pathogen avirulence (Avr) proteins either indirectly through detection of changes in their host protein targets or through direct R-Avr protein interaction. Although indirect recognition imposes selection against Avr effector function, pathogen effector molecules recognized through direct interaction may overcome resistance through sequence diversification rather than loss of function. Here we show that the flax rust fungus AvrL567 genes, whose products are recognized by the L5, L6, and L7 R proteins of flax, are highly diverse, with 12 sequence variants identified from six rust strains. Seven AvrL567 variants derived from Avr alleles induce necrotic responses when expressed in flax plants containing corresponding resistance genes (R genes), whereas five variants from avr alleles do not. Differences in recognition specificity between AvrL567 variants and evidence for diversifying selection acting on these genes suggest they have been involved in a gene-specific arms race with the corresponding flax R genes. Yeast two-hybrid assays indicate that recognition is based on direct R-Avr protein interaction and recapitulate the interaction specificity observed in planta. Biochemical analysis of Escherichia coli-produced AvrL567 proteins shows that variants that escape recognition nevertheless maintain a conserved structure and stability, suggesting that the amino acid sequence differences directly affect the R-Avr protein interaction. We suggest that direct recognition associated with high genetic diversity at corresponding R and Avr gene loci represents an alternative outcome of plant-pathogen coevolution to indirect recognition associated with simple balanced polymorphisms for functional and nonfunctional R and Avr genes.

The Melampsora lini AvrL567 avirulence genes are expressed in haustoria and their products are recognized inside plant cells.

The Linum usitatissimum (flax) L gene alleles, which encode nucleotide binding site-Leu rich repeat class intracellular receptor proteins, confer resistance against the Melampsora lini (flax rust) fungus. At least 11 different L resistance specificities are known, and the corresponding avirulence genes in M. lini map to eight independent loci, some of which are complex and encode multiple specificities. We identified an M. lini cDNA marker that cosegregates in an F2 rust family with a complex locus determining avirulence on the L5, L6, and L7 resistance genes. Two related avirulence gene candidates, designated AvrL567-A and AvrL567-B, were identified in a genomic DNA contig from the avirulence allele, whereas the corresponding virulence allele contained a single copy of a related gene, AvrL567-C. Agrobacterium tumefaciens-mediated transient expression of the mature AvrL567-A or AvrL567-B (but not AvrL567-C) proteins as intracellular products in L. usitatissimum and Nicotiana tabacum (tobacco) induced a hypersensitive response-like necrosis that was dependent on coexpression of the L5, L6, or L7 resistance gene. An F1 seedling lethal or stunted growth phenotype also was observed when transgenic L. usitatissimum plants expressing AvrL567-A or AvrL567-B (but not AvrL567-C) were crossed to resistant lines containing L5, L6, or L7. The AvrL567 genes are expressed in rust haustoria and encode 127 amino acid secreted proteins. Intracellular recognition of these rust avirulence proteins implies that they are delivered into host cells across the plant membrane. Differences in the three AvrL567 protein sequences result from diversifying selection, which is consistent with a coevolutionary arms race.

A plant gene up-regulated at rust infection sites.

Expression of the fis1 gene from flax (Linum usitatissimum) is induced by a compatible rust (Melampsora lini) infection. Infection of transgenic plants containing a beta-glucuronidase (GUS) reporter gene under the control of the fis1 promoter showed that induction is highly localized to those leaf mesophyll cells within and immediately surrounding rust infection sites. The level of induction reflects the extent of fungal growth. In a strong resistance reaction, such as the hypersensitive fleck mediated by the L6 resistance gene, there is very little fungal growth and a microscopic level of GUS expression. Partially resistant flax leaves show levels of GUS expression that were intermediate to the level observed in the fully susceptible infection. Sequence and deletion analysis using both transient Agrobacterium tumefaciens expression and stable transformation assays have shown that the rust-inducible fis1 promoter is contained within a 580-bp fragment. Homologs of fis1 were identified in expressed sequence tag databases of a range of plant species including dicots, monocots, and a gymnosperm. Homologous genes isolated from maize (Zea mays; mis1), barley (Hordeum vulgare; bis1), wheat (Triticum aestivum; wis1), and Arabidopsis encode proteins that are highly similar (76%-82%) to the FIS1 protein. The Arabidopsis homologue has been reported to encode a delta1-pyrroline-5-carboxylate dehydrogenase that is involved in the catabolism of proline to glutamate. RNA-blot analysis showed that mis1 in maize and the bis1 homolog in barley are both up-regulated by a compatible infection with the corresponding species-specific rust. The rust-induced genes homologous to fis1 are present in many plants. The promoters of these genes have potential roles for the engineering of synthetic rust resistance genes by targeting transgene expression to the sites of rust infection.