New study reveals life history trade-off — small but enhanced disease resistance in mouse ear cress (Arabidopsis thaliana)

Below is a link to a news story regarding the following empirical study published 2 days ago in the journal Nature.


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

Marco Todesco, Sureshkumar Balasubramanian, Tina T. Hu, M. Brian Traw, Matthew Horton, Petra Epple, Christine Kuhns, Sridevi Sureshkumar, Christopher Schwartz, Christa Lanz, Roosa A. E. Laitinen, Yu Huang, Joanne Chory, Volker Lipka, Justin O. Borevitz, Jeffery L. Dangl, Joy Bergelson, Magnus Nordborg & Detlef Weigel

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 1. 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 2 3 4 5. 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 6 7 8 9 10 11. 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) 12 13, 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.

A survey of A. thaliana accessions collected from the wild revealed extensive environment-dependent variation for leaf initiation rate ( Supplementary Table 1). One of the strains, Est-1, which produced leaves more slowly than the Col-0 reference strain, also developed extensive necrosis on fully expanded leaves (Fig. 1a, b). Using a recombinant inbred line (RIL) population 14, we identified single major-effect quantitative trait loci (QTL) for both leaf initiation rate and late-onset leaf necrosis (Fig. 1c), with the Est-1 alleles acting in a semi-dominant manner. We fine-mapped both QTL to the same 12-kilobase (kb) region ( Supplementary Fig. 1). We targeted the four protein-coding genes in this interval—At4g14400, At4g14410, At4g14420 and At4g14430—with artificial microRNAs (amiRNAs) 15. Knocking down At4g14400, previously identified as ACCELERATED CELL DEATH6 (ACD6) 12, suppressed late-onset necrosis and accelerated leaf initiation in Est-1 (Fig. 1a, b and Supplementary Fig. 2a), whereas downregulation of the other three genes had no visible effects. We also transformed acd6-2 loss-of-function plants in the Col-0 background with an ACD6 genomic fragment from Est-1, and found that these plants suffered from late-onset necrosis (Fig. 1a). This was not the case when we used a Col-0 genomic fragment ( Supplementary Fig. 3a). Thus, ACD6 is responsible for the leaf initiation and necrosis QTL.

ACD6 encodes a transmembrane protein with cytosolic ankyrin repeats 12 13. An ethylmethane-sulphonate-induced gain-of-function allele in Col-0, acd6-1, which carries a single amino-acid change in the transmembrane domain 12 16, is characterized by spontaneous cell death. In acd6-1 and several other so-called lesion mimic mutants, this is associated with constitutive activation of defence pathways and increased resistance to microbial infection 17, although the relationship between cell death and disease resistance is complex. Local cell death, known as the hypersensitive response, is a common consequence of pathogen recognition by genotypes with inducible immunity 18. Not all lesion-mimic mutants, however, are more resistant to pathogen attack than wild type 17, and effective disease resistance is largely uncoupled from cell death in disease no death 1 mutants 19.

Similar to acd6-1 mutants, Est-1 plants had macroscopic lesions and microscopic cell death (Fig. 1a). Furthermore, like acd6-1, PR1 and other genes mediating the response to biotic stresses were expressed much more highly in Est-1 than in Col-0 wild-type plants, and this was reproduced by transforming the Est-1 allele of ACD6 into acd6-2 loss-of-function mutants in the Col-0 background (Fig. 1d, e and Supplementary Fig. 2b). Conversely, acd6-1 mutants produced leaves more slowly than wild-type Col-0, thus mimicking Est-1 (Fig. 1f). ACD6 acts in a feed-forward loop that regulates the accumulation of salicylic acid (SA), a key molecule in pathogen defence signalling 13 16. Accordingly, conversion of SA to catechol by transgenic expression of the bacterial salicylate hydroxylase gene nahG (ref. 20) strongly attenuates acd6-1 phenotypes 12 16 21. Est-1 plants had higher SA levels than Col-0 plants, and these were strongly reduced by knocking down ACD6 (Fig. 1g). As for acd6-1, nahG expression suppressed necrosis in Est-1 ( Supplementary Fig. 4).

ACD6 RNA expression in leaves increased with age, with ACD6 levels rising earlier in Est-1 than in Col-0 ( Supplementary Fig. 2c). PR1 expression followed a similar profile only in Est-1 (Fig. 1d). PR1 levels in Est-1 were reduced after knockdown of ACD6 with the amiR-ACD6 construct ( Supplementary Fig. 2d). Conversely, PR1 expression in acd6-2 loss-of-function mutants transformed with an Est-1 genomic fragment was three orders of magnitude higher than in acd6-2 transformed with a Col-0 fragment, despite similar ACD6 levels (Fig. 1e and Supplementary Fig. 3b). The Col-0 and Est-1 proteins differ at 24 out of 670 amino acids ( Supplementary Fig. 5). Expressing ACD6 coding sequences from Est-1 under control of the Col-0 promoter was sufficient to produce an Est-1-like phenotype in acd6-2 mutants, whereas the opposite configuration did not cause any symptoms ( Supplementary Fig. 6). We conclude that changes in the protein sequence explain much of the differences in ACD6 activity between Est-1 and Col-0, which was further confirmed by expressing both alleles from a foreign promoter ( Supplementary Fig. 2e, f).

acd6-1 plants not only have necrotic lesions, but they are also small 12 16 21. Both the amiR-ACD6 transgene and nahG expression caused a marked increase, of more than 50%, in the dry weight of Est-1 leaves. The difference between wild-type and 35S::amiR-ACD6 or 35S::nahG Est-1 plants was similar to that between acd6-1 and its Col-0 parent. In contrast, the 35S::amiR-ACD6 and 35S::nahG transgenes had only minor effects on Col-0 (Fig. 2a). Similarly, introduction of the Est-1 allele, but not the Col-0 ACD6 allele, into acd6-2 loss-of-function mutants strongly reduced leaf weight ( Supplementary Fig. 3c). Altered ACD6 activity in Est-1 thus has additive effects on total biomass, by slowing the rate at which new leaves are produced and by limiting the final size of individual leaves.

acd6-1 mutants display enhanced resistance to Pseudomonas syringae pv. tomato DC3000, a hemi-biotrophic pathogen 16. We isolated a biotrophic fungus, powdery mildew Golovinomyces orontii T1, from spontaneous infections of A. thaliana in Tübingen. Est-1 was resistant to this isolate, which easily infected many other accessions including Col-0. Resistance was genetically linked to the ACD6 region ( Supplementary Fig. 1f), and knocking down ACD6 caused Est-1 to become susceptible to infection by G. orontii (Figs 2b and 3a, b). Increased susceptibility of 35S::amiR-ACD6 Est-1 plants was also seen for Golovinomyces cichoracearum UCSC1 (Fig. 3c), and for two other biotrophic pathogens: the downy mildew Hyaloperonospora arabidopsidis Noco2, an oomycete (Figs 2c and 3d and Supplementary Fig. 7), and the bacterium P. syringae DC3000 (Fig. 2d).

Variation in leaf weight associated with differences in SA content is positively correlated with several fitness-related traits, such as seed yield, in A. thaliana 22. The increased resistance to biotrophic pathogens conditioned by the Est-1 allele of ACD6 indicates that this allele can provide environment-dependent fitness advantages and may therefore not be rare, despite its negative effects on biomass. Across 96 strains from throughout the worldwide range of A. thaliana 23, 71 accessions had ACD6 alleles similar to those of Est-1 and Col-0 (Fig. 4a). The 73 strains featured a total of 141 non-synonymous substitutions, of which 67 were located in the ankyrin repeats and 17 in the predicted transmembrane domains ( Supplementary Figs 5 and 8a, b). Most of the remaining strains had an ACD6 allele, exemplified by KZ-10 that was as divergent from the Col-0 reference allele as it was from the MN47 strain of Arabidopsis lyrata (Fig. 4a, b). The relationship among the three alleles as well as At4g14390, a homologue immediately upstream of ACD6, is complex, and might involve a history of gene conversion.

Eighteen accessions shared ACD6 sequences closely related to the Est-1-like allele. All except two of these strains suffered from symptoms similar to Est-1, whereas necrosis was rare among the other 77 strains (Figs 4a and 5a, Supplementary Fig. 9a and Supplementary Table 2). These observations are consistent with the identification of the ACD6 region in a genome-wide association scan for loci causing necrosis in the same set of 96 accessions 24. Nine of the fifteen single nucleotide polymorphisms (SNPs) with the lowest P-values in the genome-wide scan were within or next to ACD6 (Fig. 4c, d). The predominance of the peak near ACD6 in the genome-wide scan demonstrates that allelic variation at this locus is the major determinant of global variation for this trait.

The group of Est-1-like alleles shared three non-synonymous substitutions in the transmembrane region (Fig. 4e and Supplementary Figs 5 and 8a, b). This region also stood out because of its excess of non-synonymous over synonymous substitutions between Col-0 and Est-1, which contrasts with this segment being highly conserved in an interspecific comparison (Fig. 4e and Supplementary Figs 8c, d and 10). An exchange of two of these non-synonymous substitutions between Est-1 and Col-0 genomic clones demonstrated that they were both necessary and sufficient for strong late-onset necrosis and activation of immune reactions (Fig. 4f and Supplementary Fig. 6b, c).

We crossed several Est-1-like accessions to the Col-0 reference strain, and confirmed in F2 populations that ACD6 co-segregated with necrosis ( Supplementary Table 2). Both reduction of SA using the 35S::nahG transgene and amiRNA-mediated knockdown of ACD6 suppressed late-onset necrosis and increased leaf biomass in several strains (Figs 2a and 5a, Supplementary Fig. 9 and Supplementary Table 2), confirming increased activity of ACD6 in these accessions.

We have shown that the Est-1-like ACD6 allele has marked effects on leaf biomass, late-onset necrosis and pathogen susceptibility. In the collection of 96 strains, we observed strong negative correlation of late-onset necrosis not only with leaf biomass, but also with resistance to G. orontii T1 and P. syringae DC3000, and with the extent to which proliferation of the aphid Myzus persicae was supported (Fig. 5b). Interestingly, whereas SNPs in the ACD6 region were strongly associated with necrosis in a genome-wide scan, associations with leaf biomass as well as G. orontii and M. persicae resistance were much weaker, and not significant at all for DC3000 growth 24 ( Supplementary Tables 3, 4 and 5). Including necrosis as a co-factor in genome-wide scans revealed additional associations outside the ACD6 region ( Supplementary Fig. 11), indicating that other factors can mask the effects of ACD6 on disease resistance.

Despite the strong sequence differentiation between ACD6 alleles, there is no obvious geographic structure to their distribution, and F ST values do not deviate from the genome-wide pattern 23 ( Supplementary Fig. 12). We also analysed a local collection of 890 A. thaliana individuals representing 202 distinct multi-locus genotypes from the Tübingen region 25. All three allele types defined by function or sequence—Col-0-like, Est-1-like and KZ-10-like—were present throughout the region, and often co-occurred ( Supplementary Fig. 13). It therefore seems that evolutionary forces maintain allelic variation at ACD6 both across the global range of A. thaliana and within local populations.

Fitness costs imposed by activation of defence have often been proposed as a possible explanation for genetic variation in disease resistance 6, and costs associated with individual genes have been detected in field trials 9 10 11. Specifically, priming of SA-related defence responses significantly increases disease resistance and plant fitness in the field 26, but reduces fitness in the absence of pathogens 22. The developmentally regulated activation of ACD6 and downstream defence components in wild A. thaliana strains carrying the hyperactive ACD6 allele (Fig. 1d and Supplementary Fig. 2c) could induce a similar primed state.

The positive association between necrosis and reduced susceptibility to many different microbes—including bacteria, oomycetes and fungi—and at least some insects is remarkable. Effectiveness of the Est-1 allele of ACD6 against such a wide range of enemies is probably due to elevated levels of SA (Fig. 1g), and to the antimicrobial compound camalexin, which is moderately increased in acd6-1 mutants 16, as well as another defence hormone, jasmonate (Fig. 2e). In this context, it is interesting that the effects of knocking down ACD6 in different accessions varied (Fig. 2a, 5a; Supplementary Fig. 9), indicating that there is a suite of genetic factors that modulate and fine-tune ACD6 activity.

The co-occurrence of functionally distinct alleles across both global and local populations of A. thaliana is consistent with this locus being under balancing selection, a pattern often seen for conventional disease resistance (R) genes 27. What sets ACD6 apart from R genes is, however, that the latter confer race-specific disease resistance, whereas ACD6 protects against a broad spectrum of unrelated enemies and predators. Unusually large benefits, in turn, might make the substantial reduction in vegetative biomass caused by ACD6 more acceptable. To put it differently, accessions with Est-1-like alleles of ACD6 seem to pursue an alternative life-history strategy, being small, but well protected, compared to other strains that are larger, but less well prepared to combat pathogens.

Methods Summary

The acd6-1 mutant 16 and the recombinant inbred line (RIL) population 14 have been described previously; the acd6-2 T-DNA insertion line was from the Salk collection 28. QTL analysis was done using the R-qtl package 29 implemented in R ( For fine mapping, we combined information from an F2 population between Col-0 and Est-1 with the heterogeneous inbred family (HIF) strategy 30.

Unless otherwise stated, plants were grown under short days (8 or 9 h light). For phenotypic assays and pathotesting, a randomized design was used. For pathogen testing with G. orontii T1, plants were grown on soil under in a greenhouse at 21–23 °C; for G. cichoracearum UCSC1, in a phytochamber at 20 °C and 60% humidity; for H. arabidopsidis Noco2, in a phytochamber at 22 °C during the day and 18 °C during the night. For P. syringae pv. tomato DC3000 and the common peach aphid Myzus persicae, plants were grown on soil in a phytochamber at 20 °C, 12 h light. Metabolites were measured using previously published methods (see Methods).

Full methods accompany this paper on the web.
Author Contributions

M.T., S.B., J.C., V.L., J.O.B., J.L.D., J.B., M.N. and D.W. conceived the study; M.T., S.B., M.B.T., M.H., P.E., C.K., S.S., C.S., C.L. and R.A.E.L. performed the experiments; M.T., S.B., T.T.H., M.B.T., Y.H., J.B., M.N. and D.W. analysed the data; and M.T., S.B. and D.W. wrote the paper with contributions from all authors.


We thank S.-W. Park and D. Klessig for the nahG clone; J. Greenberg, the NSF-supported Arabidopsis Biological Resource Centre (ABRC) and the European Arabidopsis Stock Centre (NASC) for seeds; and S. Atwell, K. Broman and Y.-L. Guo for advice. We are grateful to K. Bomblies and L. Yant for establishing the Tübingen A. thaliana collection. This work was supported by NIH NRSA fellowship F23-GM65032-1 (C.S.), an EMBO Long-Term Fellowship (S.B.), NIH grants GM62932 (J.C. and D.W.), GM057171 (J.L.D.), GM057994 (J.B.) and GM073822 (J.O.B.), NSF grants DEB-0519961 (J.B. and M.N.) and NSF MCB0603515 (J.B.), HFSPO grant RGP0057/2007-C (J.L.D. and D.W.), DFG grant LI 1317/2-1 (V.L.), the Gatsby Foundation (V.L.), the Dropkin Foundation (J.B.), the Howard Hughes Medical Institute (J.C.), Marie Curie RTN SY-STEM (D.W.), ERA-PG (BMBF) grant ARABRAS (D.W.), FP6 IP AGRON-OMICS (contract LSHG-CT-2006-037704, D.W.), a Gottfried Wilhelm Leibniz Award of the DFG (D.W.), and the Max Planck Society (D.W.).


About sociogenomics

I study behaviour and evolution. This space is about sharing with others notable (and sometimes unexpected) findings that relate to my own scientific interests and research.
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