Natural allelic variation underlying a major fitness trade-off in Arabidopsis thaliana

Abstract

Plants can defend themselves against a wide array of enemies, from microbes to large animals, yet there is great variability in the effectiveness of such defences, both within and between species. Some of this variation can be explained by conflicting pressures from pathogens with different modes of attack. A second explanation comes from an evolutionary 'tug of war', in which pathogens adapt to evade detection, until the plant has evolved new recognition capabilities for pathogen invasion. If selection is, however, sufficiently strong, susceptible hosts should remain rare. That this is not the case is best explained by costs incurred from constitutive defences in a pest-free environment. Using a combination of forward genetics and genome-wide association analyses, we demonstrate that allelic diversity at a single locus, ACCELERATED CELL DEATH 6 (ACD6), underpins marked pleiotropic differences in both vegetative growth and resistance to microbial infection and herbivory among natural Arabidopsis thaliana strains. A hyperactive ACD6 allele, compared to the reference allele, strongly enhances resistance to a broad range of pathogens from different phyla, but at the same time slows the production of new leaves and greatly reduces the biomass of mature leaves. This allele segregates at intermediate frequency both throughout the worldwide range of A. thaliana and within local populations, consistent with this allele providing substantial fitness benefits despite its marked impact on growth.

Figure 1. Identification of a natural ACD6 allele affecting growth and defence traits

a, Top, rosettes of six-week old plants. Bottom, close-up of 12^th leaf, stained with Trypan blue for dead cells. gACD6.Est is an acd6-2 mutant in Col-0 (which is morphologically normal; Supplementary Fig. 3) transformed with an Est-1 genomic fragment. b, Leaf initiation rates. c, QTL maps. Dashed black line indicates significance threshold, and ticks positions of genetic markers. d, PR1 expression in the 6^th leaf (two biological replicates each), normalized to those in 12-day old Est-1 plants. e, PR1 expression in different genotypes. f, Leaf initiation rates. g, SA content in the sixth leaf of 35-day old plants. Only wild type Est-1 was significantly different from any of the other lines (p<0.005). The 35S::amiR-ACD6 construct had no effect on SA levels in Col-0. Standard errors are indicated. Size bars in a = 1 cm (top), and 1 mm (bottom).

Figure 2. Effects of a natural ACD6 allele on leaf biomass, pathogen susceptibility and metabolite content

a, Leaf biomass. The difference between wild type and transgenic lines was significant for all accessions but Col-0 (p<0.001). b, G. orontii T1 conidiophores on four-week old plants (5 dpi). c, H. arabidopsidis Noco2 sporangiophores on 2-week old seedlings (5 dpi). d, P. syringae DC3000 growth. 35S::amiR-ACD6 did not affect susceptibility of Col-0. e, Camalexin and jasmonate concentrations. The difference between Est-1 and the other genotypes was significant (p<0.005). Standard errors are indicated.

Figure 3. Effects of a natural ACD6 allele on pathogen susceptibility

a, Infection of four-week old plants by G. orontii T1 (5 dpi). Arrows indicate fungal growth. b, Trypan blue staining of inoculated leaves. Dead plant cells (dc), hyphae (hy) and mature conidiophores (cp) are indicated. c, Infection of six-week old plants with G. cichoracearum UCSC1 (10 dpi). Note increasing severity of infection symptoms from left to right. d, Five-week old plants inoculated with H. arabidopsidis Noco 2. Trypan blue staining of the 4^th leaf (7 dpi) is shown. Hyphal growth (hy), which was seldom observed in Est-1, as well as oosporangia (os) were common in 35S::amiR-ACD6 Est-1 plants. See Supplementary Fig. 8 for adult leaves. In Col-0, many sporangiophores (sp) were seen. For both powdery and downy mildews, pathogen susceptibility and ACD6 expression levels in 35S::amiR-ACD6 lines were correlated ( see Supplementary Fig. 2a). Size bars = 1 cm in a and c, 1 mm in b and d.

Figure 4. ACD6 sequence diversity in Arabidopsis

a, Hierarchical clustering of ACD6 alleles. Col-0 and Est-1 are indicated with arrows, and Est-1-like sequences are highlighted. Yellow indicates mild, orange intermediate, and red severe late-onset necrosis. KZ-10-like alleles are gray. b, Pair-wise identity of ACD6 alleles. c, Whole-genome scan of 216,130 SNPs for association with necrosis across 96 accessions shown in a. d, Genomic region containing nine of 15 SNPs with lowest p-values. e, Polymorphism and divergence levels at ACD6 (see also Supplementary Fig. 8, 10; Supplementary Table 6). Blue lines indicate non-synonymous SNPs shared among Est-1-like alleles, and Fab-2 and Fab-4 (Supplementary Fig. 5). The two causal SNPs (see f) are indicated by asterisks, as is the acd6-1 mutation. f, Six-week old acd6-2 plants transformed with modified genomic clones of ACD6, in which two codons were swapped between Est-1 and Col-0. See also Supplementary Fig. 7. Compare to Fig. 1a and Supplementary Fig. 3a for unmutated versions. Size bars = 1 cm.

Figure 5. Correlation between late-onset necrosis, growth and defence traits

a, Late-onset necrosis in accessions with an Est-1-like ACD6 allele is suppressed by 35::amiR-ACD6, or by nahG-mediated SA depletion. See Supplementary Fig. 9 for additional accessions. b, Correlation between late-onset necrosis and different traits across 96 accessions used for genome-wide association studies. Lesioning scores reflect the range of symptoms indicated in Fig. 4a. Size bars = 2 cm for rosettes, 1 mm for micrographs.