Digital Imaging Combined with Genome-Wide Association Mapping Links Loci to Plant-Pathogen Interaction Traits

Abstract

Plant resistance to generalist pathogens with broad host ranges, such as Botrytis cinerea (Botrytis), is typically quantitative and highly polygenic. Recent studies have begun to elucidate the molecular genetic basis of plant-pathogen interactions using commonly measured traits, including lesion size and/or pathogen biomass. However, with the advent of digital imaging and high-throughput phenomics, there are a large number of additional traits available to study quantitative resistance. In this study, we used high-throughput digital imaging analysis to investigate previously poorly characterized visual traits of plant-pathogen interactions related to disease resistance using the Arabidopsis (Arabidopsis thaliana)/Botrytis pathosystem. From a large collection of visual lesion trait measurements, we focused on color, shape, and size to test how these aspects of the Arabidopsis/Botrytis interaction are genetically related. Through genome-wide association mapping in Arabidopsis, we show that lesion color and shape are genetically separable traits associated with plant disease resistance. Moreover, by employing defined mutants in 23 candidate genes identified from the genome-wide association mapping, we demonstrate links between loci and each of the different plant-pathogen interaction traits. These results expand our understanding of the functional mechanisms driving plant disease resistance.

Figure 1.

Illustration of different lesion traits. Representative images show Arabidopsis leaves infected with Botrytis illustrating the varying combinations of lesion traits observed. Lesions were observed to be more circular or more eccentric, with pointed ends growing along the midvein of the leaf. Greenness and yellowness of both areas within the lesion and in the surrounding tissue also were highly variable. The water-soaked grayish sectors represent the actual infected lesion. A, Large eccentric lesion with extensive leaf senescence. B, Moderate eccentric lesion with minimal leaf senescence. C, Small lesion with extensive leaf senescence. D, Moderate circular lesion with moderate leaf senescence. E, Small lesion with moderate leaf senescence. F, Moderate circular lesion with extensive leaf senescence.

Figure 2.

HCA of lesion traits. The heat map shows the collection of lesion measurements using the Apple517 Botrytis isolate on the collection of 96 Arabidopsis accessions (rows). The bars at the top show if the trait is linked to either size or shape of the lesion and in which spectrum it was measured, according to the legends on the right. The four traits used in this study are shown on the bottom of the plot in boldface. HCA was conducted using the Ward algorithm with z-scaling within each trait.

Figure 3.

Isolate dependency of trait correlations across the isolates. Trait correlations between each pair of traits across the 96 Arabidopsis accessions were tested via ANCOVA using each of the four different Botrytis isolates. On the left of the plots are scatterplots showing each trait correlation in each isolate, with the gray areas around the lines showing the 95% confidence interval for the prospective correlation. The dot and line color show the specific isolate tested. The diagonal contains histograms for the distributions of each specific trait across the four Botrytis isolates. The tables on the right show the P values from the ANCOVA for the trait-trait correlations, the Botrytis isolate dependency of these correlations, and the interaction of Botrytis isolate on the trait-trait correlation (Int). The estimated slopes for each trait-trait interaction for each Botrytis isolate are shown by the colored text.

Figure 4.

Lesion size GWA for four Botrytis isolates. Manhattan plots show lesion size trait GWA results as measured on four Botrytis isolates. A, Apple517. B, B05.10. C, Supersteak. D, UKRazz. The horizontal red lines show the significance threshold as estimated by permutation. The effect sizes are measured as cm² of lesion area.

Figure 5.

Venn diagram of trait-associated genes found in GWA mapping. The Venn diagram shows the candidate Arabidopsis genes found to associate with each lesion trait via GWA. The number of genes listed for each lesion trait reflects the number of genes with two or more SNPs above the 95th percentile threshold for that particular trait.

Figure 6.

Haplotype diversity effects on trait-to-genotype linkages using AT4G17010. A, Plot of z-scaled SNP effect size across all four Botrytis isolates on lesion yellowness within 1,000 bp of the AT4G17010 coding region (represented in blue blocks). The arrow indicates the transcriptional start site. The horizontal orange lines indicate the positive and negative permutation thresholds for the Botrytis isolate Apple517. The letters show the SNPs that are associated significantly with lesion yellowness in Botrytis Apple517. B, Hierarchical clustering of 95 Arabidopsis accessions based on SNPs within AT4G17010. Haplotypes are assigned into five major groups, denoted by Roman numerals. Light gray indicates that the SNP is the Col-0 allele, whereas dark gray indicates that it is the opposite allele. The SNPs are presented in their genomic order rather than the haplotype grouped structure. C, Distributions of lesion yellowness across the Arabidopsis accessions infected with Botrytis Apple517. The Arabidopsis accessions are grouped by approximate SNP haplotypes in B. The model-corrected mean value of lesion yellowness for Arabidopsis reference accession Col-0 was 0.545. Significant differences (P < 0.05) between the groups are indicated by different lowercase letters.

Figure 7.

Lesion traits affected by T-DNA insertional mutants. The Venn diagram shows the lesion traits affected significantly by each of the T-DNA knockout mutants in comparison with that in the wild-type background. The gene identifiers are located within the appropriate section of the diagram showing all the phenotypes altered by mutations in that gene.