Simon Michaeli, Marion Clavel, Esther Lechner, Corrado Viotti, Jian Wu, Marieke Dubois, Thibaut Hacquard, Benoît Derrien, Esther Izquierdo, Maxime Lecorbeiller, Nathalie Bouteiller, Julia De Cilia, Véronique Ziegler-Graff, Hervé Vaucheret, Gad Galili, and Pascal Genschik

PNAS November 5, 2019 116 (45) 22872-22883

Significance

The viral suppressor of RNA silencing P0 is known to target plant antiviral ARGONAUTE (AGO) proteins for degradation via an autophagy-related process. Here we utilized P0 to gain insight into the cellular degradation dynamics of AGO1, the major plant effector of RNA silencing. We revealed that P0 targets endoplasmic reticulum (ER)-associated AGO1 by inducing the formation of ER-related bodies that are delivered to the vacuole with both P0 and AGO1 as cargos. This process involves ATG8-interacting proteins 1 and 2 (ATI1 and ATI2) that interact with AGO1 and negatively regulate its transgene-silencing activity. Together, our results reveal a layer of ER-bound AGO1 posttranslational regulation that is manipulated by P0 to subvert plant antiviral defense.

Abstract

RNA silencing is a major antiviral defense mechanism in plants and invertebrates. Plant ARGONAUTE1 (AGO1) is pivotal in RNA silencing, and hence is a major target for counteracting viral suppressors of RNA-silencing proteins (VSRs). P0 from Turnip yellows virus (TuYV) is a VSR that was previously shown to trigger AGO1 degradation via an autophagy-like process. However, the identity of host proteins involved and the cellular site at which AGO1 and P0 interact were unknown. Here we report that P0 and AGO1 associate on the endoplasmic reticulum (ER), resulting in their loading into ER-associated vesicles that are mobilized to the vacuole in an ATG5- and ATG7-dependent manner. We further identified ATG8-Interacting proteins 1 and 2 (ATI1 and ATI2) as proteins that associate with P0 and interact with AGO1 on the ER up to the vacuole. Notably, ATI1 and ATI2 belong to an endogenous degradation pathway of ER-associated AGO1 that is significantly induced following P0 expression. Accordingly, ATI1 and ATI2 deficiency causes a significant increase in posttranscriptional gene silencing (PTGS) activity. Collectively, we identify ATI1 and ATI2 as components of an ER-associated AGO1 turnover and proper PTGS maintenance and further show how the VSR P0 manipulates this pathway.

See https://www.pnas.org/content/116/45/22872

Figure 1:

P0 and AGO1 colocalize in ER-associated bodies that are destined to the vacuole. (A) Immunoblot analysis of total, pellet (Microsomal), and supernatant (Soluble) protein preparations from XVE:P0-myc 7-d-old seedlings grown on Murashige and Skoog (MS) plates with either DMSO (−) or 10 μM β-Es (+). Blots were probed with antibodies raised against Arabidopsis AGO1, AGO4, the c-MYC tag, Luminal binding proteins BiP1/BiP2/BiP3 (BiP), and UGPase. Coomassie blue (CB) staining serves as loading control. (B) CLSM imaging of Arabidopsis root meristematic cells expressing P0-mRFP (red signal) and stained with ER-Tracker blue-white DPX (ThermoFisher; green signal). Both the merged image of the signals and an enlarged image of one of the cells are presented. Positions of the nucleus (N) and P0-mRFP labeled bodies (arrowheads) are indicated. (C) (Top and Middle) TEM imaging of ER (denoted by a dashed red line) in a root cell that underwent immunogold labeling using @AGO1 antibodies. Arrowheads mark the sites of gold particles in Top. Both Col-0 and the XVE:P0-mRFP line were treated with β-Es and ConcA. (Scale bars: 200 nm.) (Bottom) A graph showing quantification of gold particles associated with the ER, tonoplast (vac.), endosomes (end.), plasma membrane (PM), and plastid + mitochondria (pl./mit.) outer membranes. Quantification was done in samples from β-Es and ConcA treated (+B+C), or untreated, Col-0 plants. Values represent mean ± SEM. Asterisk (*) denotes statistical significance of gold particle numbers along the ER compared to the other membranes within the +B+C treated plants, P < 0.05 (Mann–Whitney U test). (D) (Top) CLSM imaging of a tobacco leaf epidermal cell transiently expressing GFP-AGO1 (AGO1), CFP-HDEL (ER), and P0-mRFP (P0). (Bottom) Enlargements of the areas bordered by yellow rectangles in Top. Bodies exhibiting both P0 and AGO1 signals are highlighted with arrowheads. (E) Immunoblot analysis of total proteins from XVE:P0-mRFP seedlings treated with β-Es for 0, 8, 24, and 48 h to induce P0-mRFP expression. Blots were probed with specific antibodies as indicated, and CB staining serves as loading control. (F) CLSM imaging of the vacuolar focal plane of root elongation zone cells from an Arabidopsis line harboring pAGO1:GFP-AGO1 and XVE:P0-mRFP and treated with β-Es and ConcA or mock (DMSO) and ConcA. Vacuole lumen is indicated (Vac).