Autophagy deficiency leads to accumulation of ubiquitinated proteins, ER stress, and cell death in Arabidopsis

Abstract

Autophagy is a homeostatic degradation and recycling process that is also involved in defense against microbial pathogens and in certain forms of cellular suicide. Autophagy has been proposed to negatively regulate plant immunity-associated cell death related to the hypersensitive response (HR), as older autophagy-deficient mutants are unable to contain this type of cell death 5 to 10 d after infection. Such spreading cell death was found to require NPR1 (nonexpressor of PR genes 1), but surprisingly did not occur in younger atg mutants. In contrast, we find that npr1 mutants are not impaired in rapid programmed cell death activation upon pathogen recognition. Furthermore, our molecular evidence suggests that the NPR1-dependent spreading cell death in older atg mutants may originate from an inability to cope with excessive accumulation of ubiquitinated proteins and ER stress which derive from salicylic acid (SA)-dependent signaling (e.g., systemic acquired resistance). We also demonstrate that both senescence and immunity-related cell death seen in older atg mutants can be recapitulated in younger atg mutants primed with ER stress. We therefore propose that the reduction in SA signaling caused by npr1 loss-of-function is sufficient to alleviate the stress levels accumulated during aging in autophagy deficient cells which would otherwise become insurmountable and lead to uncontrolled cell death.

Keywords: ER stress; age; atg; autophagy; cell death; infection; npr1; senescence; ubiquitin.

Figure 1. NPR1 is not required for the promotion of HR cell death. (A and B) Ion leakage assays of (A) 6-wk-old Col-0 WT, atg5, npr1, and atg5 npr1 plants and (B) 6-wk-old Col-0 WT, rpm1, atg2, npr1, and atg2 npr1 plants after inoculation with 1 x 10⁸ CFU mL⁻¹ Pst DC3000 (AvrRpm1). Mean and standard errors (SE) were calculated from 4 or 6 discs per treatment with 3 or 4 replicates within an experiment. Experiments were repeated with similar results. Pairwise comparisons at the last time point post infection for means vs. WT were performed with the one-way ANOVA test followed by the Holm-Šídák post-hoc test. a, P ≤ 0.05, b, P ≤ 0.01, c, P ≤ 0.0001. (C) Evaluation of tissue viability using lactophenol-trypan blue staining in leaves from Col-0 WT, npr1, atg2, atg2 npr1, and rpm1, 12 h after infection with 1 x 10⁶ CFU mL⁻¹ Pst DC3000 (AvrRpm1). Dead cells (labeled blue) are observed in leaves of all genotypes (except rpm1) treated with Pst DC3000 (AvrRpm1). No dead cells were observed in leaves infiltrated with MgCl₂ (negative control) or in rpm1 mutants which are unable to trigger HR in the presence of the AvrRpm1 effector. Size bar: 2.5 mm.

Figure 2. Loss of NPR1 function alleviates accumulation of autophagic components. Immunoblot detection of (A) ATG8A and (B) NBR1 accumulation in 3-, 6- and 9-wk-old npr1, atg2, and npr1 atg2 mutants. Coomassie Blue staining of the large subunit of RuBisCO serves as loading control.

Figure 3. Absence of NPR1 and SA responses leads to a decrease in the accumulation of ubiquitinated proteins. (A) Immunoblot detection of ubiquitin (Ubq antibody Z0458 from Dako) in 3-, 6- and 9-wk-old WT, npr1, atg2 npr1, and atg2 plants. (B and C) Immunoblot detection of (B) NBR1 and (C) ubiquitin in 7-wk-old WT, npr1, atg2 npr1, and atg2 plants, after 24 h of treatment with mock or 100 μM BTH. Amido black or Coomassie Blue staining of the large subunit of RuBisCO serve as loading controls.

Figure 4. Induction of ER stress by NPR1-dependent responses. Q-PCR of spliced BZIP60 mRNA levels in 5- and 6-wk-old Col-0 WT, npr1, atg2 npr1, and atg2 plants. Expression was normalized to wild type and is presented in a log2 scale. ACT expression was used as a standard reference.

Figure 5. Combinatory stress induced by tunicamycin and BTH leads to early senescence and uncontrolled cell death in autophagy-deficient mutants. (A) 4-wk-old Col-0 WT, npr1, atg2, and atg2 npr1 plants grown on MS plates supplemented with 0.00005% DMSO (Mock), 5 ng mL⁻¹ TM, 50 μM BTH or 5 ng mL⁻¹ TM and 50 μM BTH. Double treatment with BTH and TM decreases germination and induces early senescence in atg2 mutants but these features are rescued by NPR1 loss of function. (B) 4-wk-old WT, npr1, atg2 npr1, and atg2 leaves injected in one side with 100 ng mL⁻¹ TM (“Tunicamycin” and “AvrRpm1/Tunicamycin”) or 0.00005% DMSO (“AvrRpm1”), and then injected 5 d later with Pst DC3000 (AvrRpm1) at 2 x 10⁷ CFU mL⁻¹ (“AvrRpm1” and “AvrRpm1/Tunicamycin”) or 10 mM MgCl₂ (“Tunicamycin”), in the opposite side of the leaves. Priming of atg2 mutants with TM before infection leads to unrestricted HR cell death. Loss of NPR function in the atg2 background is sufficient to permit survival under combinatory stress and restrict HR cell death to the infection site. Pictures taken 4 d after Pst DC3000 (AvrRpm1) or MgCl₂ injections.