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

Abstract

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.

FIG. 1.—

Map of Australia showing locations of sampling sites and populations referred to in this study. Sampling locations for lineage AA and AB isolates (continental survey) are represented by open circles and closed triangles, respectively. The insert region shows locations of local populations sampled in two regions in New South Wales.

FIG. 2.—

Transient Expression of AvrP123 Genes in Flax Causes P locus Gene-Dependent Necrosis. Leaves of five different flax genotypes with different alleles of the P resistance gene locus were infiltrated with Agrobacterium tumefaciens cultures containing T-DNA plasmids encoding AvrP123 or AvrP lacking a signal peptide. Images were taken 10 days after infiltration. The variety Bison does not carry a functional P locus resistance gene, and the P, P1, P2, and P3 lines contain these resistance genes backcrossed into the variety Bison for 4–13 generations.

FIG. 3.—

Amino acid alignments for (A) AvrP123 and (B) AvrP4, with sites (clade A alleles only) under significant (P < 0.05) positive selection (M8) (shaded in dark gray). Alleles recovered from Linum marginale isolates are in black font and prefixed with Lm. Alleles recovered from L. usitatissimum isolates are in blue font and prefixed with Lu. Only those amino acids that differ from the top sequence are shown, with identical residues indicated by dots. Conserved Cysteine amino acid residues are highlighted in light gray. The names of the allelic variants used in transient transformation assays are underlined below.

FIG. 4.—

Unrooted ML trees constructed from the AvrP123 and AvrP4 sequence data. (A): AvrP123 and B: AvrP4. Bootstrap support values from 500 replicates higher than 70% are given in italics close to the nodes on top of the branches. Alleles from Melampsora lini isolates collected from Linum marginale are prefixed with Lm. Alleles from isolates collected from isolated collected from L. usitatissimum are prefixed with Lu. The number of times each allele was recovered is indicated in brackets after each allele code (with a diploid genotype inferred for each homozygote).

FIG. 5.—

Transient expression of the Melampsora lini AvrP123 and AvrP4 genes in Linum marginale causes Avr gene-dependent necrosis. (A). Leaves of a single L. marginale genotype (differential line L) displaying a necrotic response following infiltration with Agrobacterium tumefaciens cultures containing the M. lini AvrP123 alleles Lm-1 and Lm-3. (B). Leaves of a single L. marginale genotype (differential line UU) displaying a necrotic response following infiltration with A. tumefaciens cultures containing the AvrP4 alleles Lm-1, Lm-5, Lm-7, Lm-8, and Lu-2.