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.avirulence protein ͉ resistance protein P lants resist disease through a variety of preformed and induced barriers to infection. Among these is a genetically determined pathogen recognition system controlled by host resistance genes (R genes). In this gene-for-gene resistance system, plant R genes confer resistance to pathogen strains carrying corresponding avirulence (Avr) genes (so-called because their presence prevents growth on resistant plants). The recognition event involving the products of the R and Avr genes triggers host defense responses, including a localized host cell death or hypersensitive response (HR) that limits the spread of the pathogen from the infection site. Most known plant resistance proteins (R proteins) contain nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains, with the latter implicated in pathogen recognition (1). In contrast, Avr proteins are diverse, and many have pathogenicity effector functions that play important roles in enhancing infection (2). The antagonistic relationship between R and Avr genes results in coevolutionary conflict as selection favors evolution of resistance in plants and virulence in their pathogens.The Arabidopsis RPM1, RPS2, and RPS5 NBS-LRR-type R proteins confer r...

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 haustoriumspecific 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.

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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.

Thirteen alleles (L, L1 to L11, and LH) from the flax L locus, which encode Toll/interleukin-1 receptor homology-nucleotide binding site-leucine-rich repeat (TIR-NBS-LRR) rust resistance proteins, were sequenced and compared to provide insight into their evolution and into the determinants of gene-for-gene resistance specificity. The predicted L6 and L11 proteins differ solely in the LRR region, whereas L6 and L7 differ solely in the TIR region. Thus, specificity differences between alleles can be determined by both the LRR and TIR regions. Functional analysis in transgenic plants of recombinant alleles constructed in vitro provided further information: L10-L2 and L6-L2 recombinants, encoding the LRR of L2, conferred L2 resistance specificity, and an L2-L10 recombinant, encoding the LRR of L10, conferred a novel specificity. The sequence comparisons also indicate that the evolution of L alleles has probably involved reassortment of variation, resulting from accumulated point mutations, by intragenic recombination. In addition, large deletion events have occurred in the LRR-encoding regions of L1 and L8, and duplication events have occurred in the LRR-encoding region of L2.

Thirteen alleles ( L , L1 to L11 , and LH ) from the flax L locus, which encode Toll/interleukin-1 receptor homology-nucleotide binding site-leucine-rich repeat (TIR-NBS-LRR) rust resistance proteins, were sequenced and compared to provide insight into their evolution and into the determinants of gene-for-gene resistance specificity. The predicted L6 and L11 proteins differ solely in the LRR region, whereas L6 and L7 differ solely in the TIR region. Thus, specificity differences between alleles can be determined by both the LRR and TIR regions. Functional analysis in transgenic plants of recombinant alleles constructed in vitro provided further information: L10-L2 and L6-L2 recombinants, encoding the LRR of L2 , conferred L2 resistance specificity, and an L2-L10 recombinant, encoding the LRR of L10 , conferred a novel specificity. The sequence comparisons also indicate that the evolution of L alleles has probably involved reassortment of variation, resulting from accumulated point mutations, by intragenic recombination. In addition, large deletion events have occurred in the LRR-encoding regions of L1 and L8, and duplication events have occurred in the LRR-encoding region of L2 .

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 pathogenencoded 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.

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