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

Abstract

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.

Fig. 1.

Amino acid variation between AvrL567 homologs is associated with differences in recognition specificity. (A) (Left) The consensus amino acid at polymorphic positions (numbered vertically above the consensus line) in the AvrL567 proteins is shown above the individual sequences with identical residues indicated by dots. (Right) The columns indicate whether a necrotic response was observed when these proteins were expressed in flax lines containing L5, L6, L7, or the recombinant L6L11RV gene. ++ indicates a very strong necrotic response observed within 4 days; + indicates necrosis observed within 10 days; +/− indicates a chlorotic response observed after 10 days; − indicates no response observed. ∗, A slight chlorotic response was observed for AvrL567-E on L6 in some but not all assays, suggesting very weak recognition. (B) Leaves of near-isogenic flax lines containing the L9, L5, L6, or L7 R genes or transgenic flax plants containing L6L11RV were infiltrated with Agrobacterium cultures containing T-DNA expression vectors encoding the predicted 127-aa mature AvrL567-D, AvrL567-F, AvrL567-J, or AvrL567–L proteins under the control of the cauliflower mosaic virus 35S promoter. Images were prepared 10 days after infiltration.

Fig. 2.

AvrL567 proteins interact specifically with the corresponding R proteins in yeast. (A) Growth of yeast strain HF7c expressing both GAL4-AD and GAL4-BD fusion proteins on minimal media lacking histidine. (Upper) Growth of strains expressing AD::AvrL567 fusion proteins (A to L) with BD alone (-ve), or BD::L5, BD::L6, or BD::L6L11RV fusion proteins. (Lower) Growth of strains expressing the reversed fusions, i.e., BD::AvrL567 proteins (A to L) with AD alone (-ve), AD::L5, AD::L6, or AD::L6L11RV. Growth indicates expression of the HIS3 reporter gene as a result of interaction between the GAL4-AD and GAL4-BD fusion proteins. (B) Yeast strain SFY526 expressing the GAL4 fusion proteins was assayed for β-galactosidase activity. (Upper) Activity of strains expressing AD::AvrL567 proteins (A to L) with BD alone (-ve), BD::L5, BD::L6, or BD::L6L11RV. (Lower) Activity of strains expressing the reversed fusions. (C) β-Galactosidase activity of yeast strain SFY526 expressing GAL4-BD fused to modified L6 proteins containing a K271M (L6ΔP-loop) or a D541V (L6-MHV) mutation along with AD alone (-ve), AD::AvrL567-A, or AD::AvrL567-D. (D) Protein extracts from yeast strain SFY526, and SFY526 expressing AD::L5, AD::L6, AD::L6-D541V (MHV), AD::L6-K271M (P-loop) ADand::L6L11 (Left) or AD::AvrL567 (A to L; Right) were analyzed by immunoblotting with anti-hemagglutinin (HA) or anti-GAL4-AD mAbs. Positions and sizes of protein molecular mass standards are indicated.

Fig. 3.

AvrL567-A, AvrL567-C, and AvrL567–D proteins exhibit similar structural characteristics. (A) CD spectra of purified recombinant AvrL567-A, AvrL567-C, and AvrL567-D proteins. The spectra indicate that the proteins consist mainly of β-sheet secondary structure. (B) AvrL567 proteins were subjected to limited proteolysis with endopeptidase Glu-C (preferentially cleaves C terminal to glutamic acid residues) and chymotrypsin (cleaves C terminal to bulky hydrophobic residues). Treatment with either protease yielded a protected fragment of ≈14 kDa, which remained stable over a period of 24 h (data not shown). N-terminal sequencing and mass spectrometry were used to identify the protected fragments. The observed cleavage sites in the mature AvrL567 protein are indicated by arrows: red, chymotrypsin cleavage sites; black, Glu-C cleavage sites. No cleavage in the C-terminal region was observed.