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. 2018 Sep 20;14(9):e1007628.
doi: 10.1371/journal.pgen.1007628. eCollection 2018 Sep.

Modulation of ACD6 Dependent Hyperimmunity by Natural Alleles of an Arabidopsis Thaliana NLR Resistance Gene

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Free PMC article

Modulation of ACD6 Dependent Hyperimmunity by Natural Alleles of an Arabidopsis Thaliana NLR Resistance Gene

Wangsheng Zhu et al. PLoS Genet. .
Free PMC article

Abstract

Plants defend themselves against pathogens by activating an array of immune responses. Unfortunately, immunity programs may also cause unintended collateral damage to the plant itself. The quantitative disease resistance gene ACCELERATED CELL DEATH 6 (ACD6) serves to balance growth and pathogen resistance in natural populations of Arabidopsis thaliana. An autoimmune allele, ACD6-Est, which strongly reduces growth under specific laboratory conditions, is found in over 10% of wild strains. There is, however, extensive variation in the strength of the autoimmune phenotype expressed by strains with an ACD6-Est allele, indicative of genetic modifiers. Quantitative genetic analysis suggests that ACD6 activity can be modulated in diverse ways, with different strains often carrying different large-effect modifiers. One modifier is SUPPRESSOR OF NPR1-1, CONSTITUTIVE 1 (SNC1), located in a highly polymorphic cluster of nucleotide-binding domain and leucine-rich repeat (NLR) immune receptor genes, which are prototypes for qualitative disease resistance genes. Allelic variation at SNC1 correlates with ACD6-Est activity in multiple accessions, and a common structural variant affecting the NL linker sequence can explain differences in SNC1 activity. Taken together, we find that an NLR gene can mask the activity of an ACD6 autoimmune allele in natural A. thaliana populations, thereby linking different arms of the plant immune system.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Genetic background dependence of ACD6-Est effect.
(A) Variation of necrosis in 54 natural accessions (n = 6 each) with ACD6-Est alleles. Broad-sense heritability H2 is 0.89. (B) Accumulation of ACD6 mRNA in various genetic backgrounds (n = 3), as measured by qRT-PCR. Asterisks indicate significant differences in pairwise Student’s t-tests (p<0.0001; Holm adjusted). Letters indicate significantly different groups (p<0.0001; post hoc Tukey’s HSD). (C) Representative non-transgenic and ACD6 transgenic lines at 5 weeks of age in Col-0 and acd6-2 backgrounds. Est-1, Pro-0 and Rmx-A180 are shown for comparison on the left. Scale bar, 10 mm.
Fig 2
Fig 2. ACD6-dependent suppression of salicylic acid accumulation and flg22 responses in Pro-0.
(A) SA levels in lines with different ACD6 alleles, n = 10 per line. Asterisks indicate pairwise Student’s t-test results (p<0.0001; Holm adjusted). Letters indicate significantly different groups (p<0.0001; post hoc Tukey’s HSD). (B) ACD6 and PR1 expression in different T1 individuals (n = 4), normalized to Col-0. Asterisks indicate significance in pairwise Student’s t-tests (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Holm adjusted). Letters indicate significantly different groups (p<0.0001; post hoc Tukey’s HSD). (C) H2O2 production in response to flg22 (n = 4). Error bars represent standard errors. This experiment was repeated four times with similar results.
Fig 3
Fig 3. Genetic analysis of ACD6 modifiers in natural accessions.
(A) Necrosis QTL mapping in three F2 populations. Dashed lines indicate LOD score significance thresholds of p<0.05 (from 1,000 permutations). (B) Representative phenotypes of transformants expressing genomic SNC1 fragments. Scale bar, 10 mm. (C) Necrosis in transgenic Pro-0 plants. The effects of SNC1-Est-1 and SNC1-Pro-0 transgenes were significantly different (chi-square test, p<0.001). (D) Growth of Pst DC3000 after infection at OD600 = 0.0001. Bacterial growth in Pro-0 [gSNC1-Pro-0] was significantly different from either Pro-0 [gSNC1-Est-1] or nontransgenic Pro-0 on day 3. Asterisks indicate significance in pairwise Student’s t-tests against Pro-0 (*p<0.05, ***p<0.001, **** p<0.0001; Holm adjusted). Letters indicate significantly different groups (p<0.0001; post hoc Tukey’s HSD). L1/L2 designates two independent transgenic lines for both constructs.
Fig 4
Fig 4. Correlation between SNC1 alleles and ACD6-Est-1 effects in natural accessions.
(A) Phylogeny of 136 RPP4/5 and SNC1 proteins from 65 accessions. Bootstrap values over 60% are indicated. The SNC1 clade is highlighted in color, with single NL linker (SNC1-sNL) genes green and duplicated NL linker (SNC1-dNL) genes brown. Arrows highlight five accessions with two SNC1 homologs. (B) Diagrams of SNC1 proteins with single and duplicated NL linkers. Gene IDs for five accessions with two SNC1 homologs on the right. (C) Accessions with ACD6-Est-like alleles; SNC1 types indicated below. Scale bar, 10 mm.
Fig 5
Fig 5. Contribution of NL linker variation to SNC1 activity.
(A) Distribution of necrosis in Col-0 T1 transformants expressing gSNC1-Pro-0 with different NL linkers. Asterisks indicate pairwise chi-square test results (***p<0.001, ****p<0.0001; Holm adjusted). (B) Left, ion leakage, an indicator of cell death, in N. benthamiana leaves transiently transformed with different gSNC1 transgenes 4 d after Agrobacterium infiltration. Asterisks indicate significance in pairwise Student’s t-tests against Est-1 (***p<0.001, ****p<0.0001; Holm adjusted). Letters indicate significantly different groups (p<0.0001; post hoc Tukey’s HSD). The leaf samples were collected from six independent plants. Right, image of a representative 7-day old leaf after infiltration.

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Grant support

This work was supported by European Research Council, Advanced Grants, IMMUNEMESIS, and the Max Planck Society. The NLR RenSeq database was generated with generous support from the Gordon and Betty Moore Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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