Fig 1.
Mutants with runaway cell death accumulate DNA damage in uninfected conditions.
pub13 and vad1 mutants have more DNA damage than Col-0 or dnd1. (A) Representative images of comets and (B) % tail DNA quantification of the genotypes. Values of 3 biological replicates made of pools of different individuals (at least 50 comets scored per biological replicate). Bars marked with different letters are statistically different (P≤ 0.01) among samples according to a Holm-Sidak multiple comparison test. (C) Immunoblot of histone extraction from Col-0, dnd1 and vad1 probed with anti γ-H2AX antibody. Unspecific band was used as loading control. (D) Quantification of the immunoblot of (C) γ-H2AX analysis normalized to input and to Col-0 (set to 100,Values are mean ± SD of 2 biological replicates).
Fig 2.
DNA damage accumulation in the camta 3 mutant is dependent on NLR signalling.
Accumulation of DNA damage in camta 3–1 is dependent on the NLR signalling component EDS1 and on the NLR DSC2. (A) Representative pictures of comets and (B) % tail DNA quantification of the genotypes. Values are of 3 biological replicates made of pools of different individuals (at least 50 comets scored per biological replicate). Bars marked with different letters are statistically different (P≤ 0.01) among samples according to a Holm-Sidak multiple comparison test. (C) Immunoblot of histone extraction from Col-0, camta3 and camta3 expressing DN-DSC2 probed with anti γ-H2AX antibody. Unspecific band was used as loading control. (D) Quantification of the immunoblot of (C) γ-H2AX analysis normalized to input and to Col-0 (set to 100, values are mean ± SD of 2 biological replicates).
Fig 3.
SA analogues BTH and INA do not induce significant accumulation of DNA damage.
Col-0 plants treated with SA, INA or BTH do not display significant DNA damage accumulation when compared to untreated plants. (A) Representative pictures of comets and (B) % tail DNA quantification of the conditions described. Values of 3 biological replicates made of pools of different individuals (at least 50 comets scored per biological replicate). Bars marked with different letters are statistically different (P≤ 0.01) among samples according to a Holm-Sidak multiple comparison test. (C) Immunoblot of histone extraction from Col-0, camta3 and Col-0 + 1mM SA probed with anti γ-H2AX antibody. Ponceau staining was used as loading control. (D) Quantification of the immunoblot of (C) γ-H2AX analysis normalized to input and to Col-0 (set to 100) (Values are mean ± SD of 2 biological replicates).
Fig 4.
ETI activation leads to DNA damage accumulation even in the absence of pathogens.
Recognition of a single effector (avrRPM1) is sufficient to induce DNA damage accumulation. (A and B) Col-0 accumulated more DNA damage than rpm1-3 mutants infected with P. fluorescens harboring avrRPM1. (C and D) In planta expression of avrRPM1 under control of a DEX inducible promotor is sufficient to cause DNA damage (8h after treatment). (A and C) Representative pictures of comet assays and (B and D) Tail DNA % quantification of the genotypes and conditions described. Values of 3 biological replicates made of pools of different individuals (at least 50 comets scored per biological replicate). Bars marked with different letters are statistically different (P≤ 0.01) among samples according to a Holm-Sidak multiple comparison test. (E) Immunoblot of samples of plants sprayed with DEX after the given time points probed with anti γ-H2AX antibody. (F) Quantification of the immunoblot of (C) γ-H2AX analysis normalized to input and to 0h sample (set to 100) (Values are mean ± SD of 2 biological replicates).
Fig 5.
sni1 autoimmune phenotype is dependent of EDS1.
(A) picture of 5 week-old plants grown under short day conditions displaying partial rescue of sni1 dwarfism in sni1 eds1 (8h days). (B) Trypan blue staining of 2 week-old sni1, sni1 eds1-2 and eds 1–2 plants showed that run-away cell death in sni1 is dependent on EDS1. (C) PR1 relative transcript accumulation in sni1 was abrogated in the sni1 eds1-2 double mutant. Results, normalized to UBQ10 and relative to Col-0, are shown as mean ± SD of 3 biological replicates.
Fig 6.
DNA damage accumulation in sni1 is caused by autoimmunity.
(A and B) DNA damage accumulation in sni1, sni1 eds1-2 and eds1-2. (A) Representative pictures of comets and (B) % tail DNA quantification of the genotypes. Values of 3 biological replicates made of pools of different individuals (at least 50 comets scored per biological replicate). Bars marked with different letters are statistically different (P≤ 0.01) among samples according to a Holm-Sidak multiple comparison test.
Fig 7.
DNA Damage Repair shutdown is dependent on ETI signalling components.
(A to D) RAD51 accumulates in an immunity dependent manner. (A) sni1 displays enhanced accumulation of RAD51 which is reduced in sni1 eds1-2. Immunoblot of total protein extracts from eds1-2, sni1, sni1 eds1-2 probed with anti RAD51 antibody. An unspecific band was used as loading control. (B) Quantification of the immunoblot of (B) RAD51 normalized to input and to Col-0 (set to 100) (Values are mean ± SD of 3 biological replicates). (C) ETI activation causes RAD51 degradation by proteases with Caspase 3-like activity. Protein extracts from plants subjected to the conditions given were probed with anti RAD51 antibody. Unspecific band was used as loading control. (D) Quantification of the immunoblot of (C) RAD51 normalized to input and to Col-0 infected at 2h (set to 100) (Values are mean ± SD of 3 biological replicates). (E and F) DDR genes are downregulated during HR PCD, (E) downregulation of DDR genes in camta 3–1 and sni1 is dependent of EDS1. (F) vad1 and pub 13, but not dnd1, display DDR gene downregulation. Gene expression was normalized to UBQ10 and is relative to Col-0 (values represent the average ± SD of 3 biological replicates).
Table 1.
Primers list for qPCR and genotyping.