The novel avirulence effector AlAvr1 from Ascochyta lentis mediates host cultivar specificity of ascochyta blight in lentil

Abstract

Ascochyta lentis is a fungal pathogen that causes ascochyta blight in the important grain legume species lentil, but little is known about the molecular mechanism of disease or host specificity. We employed a map-based cloning approach using a biparental A. lentis population to clone the gene AlAvr1-1 that encodes avirulence towards the lentil cultivar PBA Hurricane XT. The mapping population was produced by mating A. lentis isolate P94-24, which is pathogenic on the cultivar Nipper and avirulent towards Hurricane, and the isolate AlKewell, which is pathogenic towards Hurricane but not Nipper. Using agroinfiltration, we found that AlAvr1-1 from the isolate P94-24 causes necrosis in Hurricane but not in Nipper. The homologous corresponding gene in AlKewell, AlAvr1-2, encodes a protein with amino acid variation at 23 sites and four of these sites have been positively selected in the P94-24 branch of the phylogeny. Loss of AlAvr1-1 in a gene knockout experiment produced a P94-24 mutant strain that is virulent on Hurricane. Deletion of AlAvr1-2 in AlKewell led to reduced pathogenicity on Hurricane, suggesting that the gene may contribute to disease in Hurricane. Deletion of AlAvr1-2 did not affect virulence for Nipper and AlAvr1-2 is therefore not an avirulence gene for Nipper. We conclude that the hemibiotrophic pathogen A. lentis has an avirulence effector, AlAvr1-1, that triggers a hypersensitive resistance response in Hurricane. This is the first avirulence gene to be characterized in a legume pathogen from the Pleosporales and may help progress research on other damaging Ascochyta pathogens.

Keywords: Ascochyta lentis; Lens culinaris; Didymellaceae; Dothideomycete; Pleosporales; avirulence; hemibiotroph.

FIGURE 1

Disease scores for a segregating Ascochyta lentis mapping population. (a) Normalized disease scores for PBA Hurricane XT and Nipper infected with A. lentis isolates AlKewell and P94‐24 (boxed), and biparental progeny at 14 days after inoculation. Error bars show the standard error for four biological replicates. A horizontal line at 0.1 represents an arbitrary threshold below which strains were considered to be avirulent. (b) Distribution histogram of normalized disease scores for biparental progeny. (c) Linear regression analysis of biparental progeny for normalized Nipper versus Hurricane disease scores

FIGURE 2

Quantitative trait locus (QTL) analysis by linear mixed model for ascochyta blight disease scores for the susceptible control lentil cultivar ILL6002, and for Nipper and PBA Hurricane XT. Logarithm of odds (LOD) thresholds for Nipper (dashed line) and Hurricane (dotted line) are shown. A significant QTL with LOD score of 17.0 (LOD threshold, p < 0.005 = 4.71) was found on Ascochyta lentis chromosome 3

FIGURE 3

Gene features for Ascochyta lentis g2688. (a) Genomic context of the g2688 gene on A. lentis chromosome 3 (red box). Genes (green) and coding sequences (CDS) (yellow) are indicated along the region of the chromosome. The GC content graph below the chromosome shows GC content (green) and AT content (blue). (b) Alignments for AlKewell and P94‐24 forms of g2688, respectively, and corresponding protein sequences. Nucleotide identity and polymorphisms between isolates are shown above the alignments as green and white bands, respectively. cDNA and protein diagrams for each isolate are shown below as grey bars on which nucleotide and amino acid changes are shown with nucleotide and amino acid‐specific colours. Signal peptides are indicated with a green box. Cysteine residues are indicated with blue boxes and four amino acid sites under positive selection are indicated with red boxes. Asterisks at around the 450 bp position show amino acid residues potentially mutated as a consequence of repeat‐induced point mutation

FIGURE 4

Functional characterization of P94‐24 and AlKewell g2688 isoforms, labelled as g2688‐P and g2688‐K, respectively, by agroinfiltration in different lentil cultivars. Representative images of agroinfiltrated leaves from three independent experiments, each with three technical replicates, observed at 5 days postinfiltration

FIGURE 5

Gene expression of AlAvr1‐2 in AlKewell (a) and AlAvr1‐1 in P94‐24 (b) during infection of lentil PBA Hurricane XT and Nipper. The expression level of AlAvr1‐1 and AlAvr1‐2 in respective wild‐type (WT) isolates during the course of infection was examined using reverse transcription‐quantitative PCR. Quantitative weight measurement was based on a standard curve, and the level of gene expression was normalized to actin gene expression. Error bars represent standard error of the mean from three biological replicates. Data is plotted on a log₁₀ scale. Results not connected by the same letter are significantly different (Student's t test, p < 0.01 (a) and p < 0.001 (b))

FIGURE 6

Pathogenicity of AlAvr1 gene knockout strains. (a) Infection of PBA Hurricane XT and Nipper lentil with AlKewell wild‐type (WT) and the AlAvr1‐2 knockout mutant ΔAlAvr1‐2 in the AlKewell background (b) P94‐24 WT and the AlAvr1‐1 knockout mutant ΔAlAvr1‐1 in the P94‐24 background. Results are reported as the mean of % leaf area damage from nine individual plants scored 14 days postinfection and the data were square‐root‐transformed. Results not connected by the same letter are significantly different (Tukey HSD test, p < 0.0001)