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.

https://www.pnas.org/content/pnas/103/23/8888.full.pdf

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

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 .

Identification of regions in alleles of the flax rust resistance gene L that determine differences in gene-for-gene specificity

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.

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

The M rust resistance gene from flax was cloned after two separate approaches, an analysis of spontaneous M mutants with an L6 gene-derived DNA probe and tagging with the maize transposon Activator, independently identified the same gene. The gene encodes a protein of the nucleotide binding site leucine-rich repeat class and is related (86% nucleotide identity) to the unlinked L6 rust resistance gene. In contrast to the L locus, which contains a single gene with multiple alleles, approximately 15 related genes occur at the complex M locus, with only one encoding the M resistance specificity. The M protein contains two direct repeats of 147 and 149 amino acids in the C-terminal part of the leucine-rich region. Three mutant alleles of M encoding a product containing a single repeat unit of 154 amino acids were isolated. The mutant DNA sequences probably occurred by unequal intragenic exchange in the coding region of the repeats. The recombinant alleles lost M resistance and gained no detectable new resistance specificity.

Inactivation of the flax rust resistance gene M associated with loss of a repeated unit within the leucine-rich repeat coding region.

At least six rust resistance specificities (P and P1 to P5) map to the complex P locus in flax. The P2 resistance gene was identified by transposon tagging and transgenic expression. P2 is a member of a small multigene family and encodes a protein with nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains and an N-terminal Toll/interleukin-1 receptor (TIR) homology domain, as well as a C-terminal non-LRR (CNL) domain of ∼150 amino acids. A related CNL domain was detected in almost half of the predicted Arabidopsis TIR-NBS-LRR sequences, including the RPS4 and RPP1 resistance proteins, and in the tobacco N protein, but not in the flax L and M proteins. Presence or absence of this domain defines two subclasses of TIR-NBS-LRR resistance genes. Truncations of the P2 CNL domain cause loss of function, and evidence for diversifying selection was detected in this domain, suggesting a possible role in specificity determination. A spontaneous rust-susceptible mutant of P2 contained a G→E amino acid substitution in the GLPL motif, which is conserved in the NBS domains of plant resistance proteins and the animal cell death control proteins APAF-1 and CED4, providing direct evidence for the importance of this motif in resistance gene function. A P2 homologous gene isolated from a flax line expressing the P resistance specificity encodes a protein with only 10 amino acid differences from the P2 protein. Chimeric gene constructs indicate that just six of these amino acid changes, all located within the predicted β-strand/β-turn motif of four LRR units, are sufficient to alter P2 to the P specificity.

Six Amino Acid Changes Confined to the Leucine-Rich Repeat β-Strand/β-Turn Motif Determine the Difference between the P and P2 Rust Resistance Specificities in Flax

A list with information about named host genes controlling resistance to rust in both cultivated and wild flax has been compiled. These will be useful for genetic, physiological and biochemical research as well as breeding for resistance. Information regarding mutation, temperature sensitivity and the effect of inhibitor/avirulence gene interaction on expression of certain host genes is included.

M. R. Islam

Papers

A Compendium on Host Genes in Flax Conferring Resistance to Flax Rust