Degradation of the antiviral component ARGONAUTE1 by the autophagy pathway

Abstract

Posttranscriptional gene silencing (PTGS) mediated by siRNAs is an evolutionarily conserved antiviral defense mechanism in higher plants and invertebrates. In this mechanism, viral-derived siRNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation of the corresponding viral RNAs. In Arabidopsis, a key component of RISC is ARGONAUTE1 (AGO1), which not only binds to siRNAs but also carries the RNA slicer activity. At present little is known about posttranslational mechanisms regulating AGO1 turnover. Here we report that the viral suppressor of RNA silencing protein P0 triggers AGO1 degradation by the autophagy pathway. Using a P0-inducible transgenic line, we observed that AGO1 degradation is blocked by inhibition of autophagy. The engineering of a functional AGO1 fluorescent reporter protein further indicated that AGO1 colocalizes with autophagy-related (ATG) protein 8a (ATG8a) positive bodies when degradation is impaired. Moreover, this pathway also degrades AGO1 in a nonviral context, especially when the production of miRNAs is impaired. Our results demonstrate that a selective process such as ubiquitylation can lead to the degradation of a key regulatory protein such as AGO1 by a degradation process generally believed to be unspecific. We anticipate that this mechanism will not only lead to degradation of AGO1 but also of its associated proteins and eventually small RNAs.

Fig. 1.

P0-mediated degradation of AGO1 is blocked by autophagy inhibitors. AGO1 degradation kinetics were performed on 7-d-old XVE-P0^BW-myc seedlings treated with β-estradiol (5 μM) for P0-myc induction. Autophagy was inhibited in its last steps using E64d (20 μM) (A) and AGO1, P0-myc, and CDC2 (loading control) protein accumulation levels were assayed by Western blot on a 24-h period. In a similar manner, AGO1 mRNA (B) and miR168 accumulation (C) was assayed by Northern blot analyses along P0-mediated degradation of AGO1 in presence or in absence of E64d (20 μM). Loading controls are methylene blue staining of the membrane for mRNA and U6 for small RNA blots. (D) Autophagy was inhibited in its first steps using the specific PI-3-kinase class III inhibitor 3-MA (5 mM) and AGO1, P0-myc, and CDC2 (loading control) protein accumulation levels were assayed by Western blot on a 24-h period. (E) Coimmunoprecipitation of AGO1 and SCF^P0. XVE-P0^BW-myc seedlings were treated with β-estradiol (5 μM) for P0-myc induction and E64d (20 μM) for at least 6 h before protein extraction. Plant extract were immunoprecipitated with an anti-AGO1 antibody and with normal rabbit serum (NRS). IP fractions were submitted to Western blot analysis using antibodies raised against the myc tag for P0 detection and against CUL1, AGO1, and ATG8a. (F) Ubiquitylation status of AGO1 was determined by Western blot analysis of IP fractions using an antibody specifically raised against K63-Ub. (G) Inhibition of SCF activity prevents P0-mediated degradation of AGO1. Seven-day-old XVE-P0^BW-myc seedlings were pretreated with MLN-4924 (25 μM) for 3 h before P0-myc induction with β-estradiol (5 μM). The accumulation level of AGO1, P0-myc, CUL1, and CDC2 (loading control) was assayed by Western blot 24 h after P0 induction. Anti-CUL1 antibody detects two bands, the upper one corresponding to the NEDD8/RUB1-modified form of CUL1.

Fig. 2.

Subcellular localization of AGO1 along its degradation process. (A) The pAGO1:GFP-AGO1 construct complements ago1-27 allele phenotype. (B) Subcellular localization of functional GFP-AGO1 assayed by confocal microscopy. Seven-day-old seedlings were transferred from MS-agar plates to liquid MS medium supplemented with the indicated drugs and observed after overnight incubation (16–18 h). In the root tip of XVE-P0^BW/GFP-AGO1 reporter lines GFP-AGO1 is localized exclusively in the cytoplasm of cells (Left). After P0 induction, the GFP-AGO1 signal decreases with a nonhomogenous pattern from cell to cell and is relocalized in vesicular-shaped structures (Middle). When P0 induction is combined with E64d (20 μM) treatment, GFP-AGO1 is stabilized and massively accumulates in these vesicular-shaped structures (Right). These speckles colocalize with acidic vesicles labeled with LysoTracker Red DND-99 (LyTr) (C) and their formation is significantly reduced if P0 induction and E64d treatment are combined with 3-MA (5 mM) (D). (Scale bars: 10 μm.)

Fig. 3.

P0 expression leads to an accumulation of vacuolar inclusions containing GFP-AGO1. (A) Root-tip cells in which P0 expression was induced with β-estradiol (+βestr) for 12–16 h display electron-dense inclusions in several vacuole-like structures (arrows), and in mock treated cells (B) vacuoles do not contain this kind of inclusions. (A and B, scale bars: 2 μm). (C) The vacuolar identity of the compartments containing the inclusions was confirmed by immunodetection of the vacuolar pyrophosphatase (V-PPase); nanogold particles coupled to secondary antibodies uniformly label the limiting membrane (arrowheads). (D–F) Immunogold labeling of GFP-AGO1 after P0 induction and additional treatment with the protease inhibitor E64d (+βestr+E64d) revealed that AGO1 is present on membranous structures within the inclusions (arrows). V, vacuoles. (C–F, scale bars: 200 nm.)

Fig. 4.

P0-mediated degradation of AGO1 is compromised in amsh3-1 mutant and in TOR-overexpressing plants and the endogenous pathway for AGO1 degradation also relies on autophagy. (A) P0-dependant degradation of AGO1 in XVE-P0^BW-myc/amsh3-1 line. (Left) AGO1 protein accumulation level 24 h after P0 induction (10 μM β-estradiol) on 11-d-old seedlings. (Right) AGO1 accumulation level in 10-d-old seedlings that have been germinated and grown on MS-agar dish containing 10 μM β-estradiol. (B) AGO1 degradation kinetics performed on 7-d old XVE-P0^BW-myc and XVE-P0^BW-myc/G548 seedlings treated with β-estradiol (5 μM) for P0-myc induction. Because P0 induction is delayed in the XVE-P0^BW-myc/G548 line, we extended this kinetic to 24 h. AGO1, P0-myc, and CDC2 (loading control) protein contents were assayed by Western blot. (C) E64d treatment (20 μM for 24 h) on wild-type Col-0 seedling leads to a higher accumulation of AGO1 protein. (D) Mutants affected in miRNA maturation and production pathways show reduced level of AGO1. (Upper) AGO1 protein accumulation in mutants and wild-type controls assayed by Western blot using the anti-AGO1 antibody. Coomassie blue staining is given as loading control. (Lower) AGO1 mRNA accumulation in each mutant assayed by quantitative RT-PCR. (E) The hen1-1 seedlings treated with E64d (20 μM) show AGO1 protein reaccumulation. AGO1 and CDC2 (loading control) protein contents were assayed by Western blot at the indicated time point. (F) Model for AGO1 turnover in a viral and nonviral context.