Monitoring Autophagy in Rice With GFP-ATG8 Marker Lines

Abstract

Autophagy is a conserved intracellular trafficking pathway for bulk degradation and recycling of cellular components in eukaryotes. The hallmark of autophagy is the formation of double-membraned vesicles termed autophagosomes, which selectively or non-selectively pack up various macromolecules and organelles and deliver these cargoes into the vacuole/lysosome. Like all other membrane trafficking pathways, the observation of autophagy is largely dependent on marker lines. ATG8/LC3 is the only autophagy-related (ATG) protein that, through a covalent bond to phosphatidylethanolamine (PE), associates tightly with the isolation membrane/pre-autophagosomal structure (PAS), the growing phagophore, the mature autophagosome, and the autophagic bodies. Therefore, fluorescent protein (FP)-tagged ATG8 had been widely used for monitoring autophagosome formation and autophagic flux. In rice (Oryza sativa), FP-OsATG8 driven by Cauliflower mosaic virus (CaMV) 35S promoter had been used for imaging autophagosome and autophagic bodies. Here, we constructed three vectors carrying GFP-OsATG8a, driven by 35S, ubiquitin, and the endogenous ATG8a promoter, individually. Then, we compared them for their suitability in monitoring autophagy, by observing GFP-ATG8a puncta formation in transiently transformed rice protoplasts, and by tracking the autophagic flux with GFP-ATG8 cleavage assay in rice stable transgenic lines. GFP-Trap immunoprecipitation and mass spectrometry were also performed with the three marker lines to show that they can be used reliably for proteomic studies. We found out that the ubiquitin promoter is the best for protoplast imaging. Transgenic rice seedlings of the three marker lines showed comparable performance in autophagic flux measurement using the GFP-ATG8 cleavage assay. Surprisingly, the levels of GFP-ATG8a transcripts and protein contents were similar in all marker lines, indicating post-transcriptional regulation of the transgene expression by a yet unknown mechanism. These marker lines can serve as useful tools for autophagy studies in rice.

Keywords: ATG8; autophagic flux; autophagy; post-transcriptional regulation; rice.

Figure 1

Transient expression of constructs carrying GFP-OsATG8a. (A) Schematic representation of the GFP-OsATG8a fusion gene constructs. Three promoters—the CaMV35S promoter, the ubiquitin (UBQ10) promoter, and the endogenous (OsATG8a) promoter—are used for the constructs. (B) LSCM of tobacco leaf epidermal cells transiently expressing GFP and GFP-OsATG8a. (C) Transient expression of GFP and GFP-OsATG8a in rice protoplasts. Puncta representing autophagosomes are indicated by yellow arrowheads in (B,C). Bar = 10 μm in (B,C).

Figure 2

Visualization of autophagic body accumulation in rice protoplasts. Rice protoplasts expressing GFP-OsATG8a driven by (A) 35S promoter, (B) UBQ10 promoter, and (C) OsATG8a promoter were observed with LSCM after treatment with 250 mM NaCl, or 50 μM E-64d, or NaCl plus E-64d for 30 min. Accumulation of GFP signals, indicative of autophagosome and autophagic bodies, can be seen in protoplasts treated with NaCl plus E-64d. Bar = 10 μm in (A,B); 5 μm in (C).

Figure 3

Measuring autophagic flux in GFP-OsATG8a marker lines. The level of autophagic flux is represented by the difference of the GFP-ATG8 cleavage between control and treatment. GFP-ATG8 cleavage was calculated as the ratio of the free GFP band (27 kD) to the total GFP signal in the lane (40kD plus 27 kD), then normalized to the respective control (no treatment, no E-64d or ConA; set as 1.00). A color scale (Blue for 0, white for 1, and red for 2) was used to illustrate the semi-quantified values of GFP-ATG8 cleavage. Coomassie Brilliant Blue (CBB) staining of RuBisCO large subunit was used as loading control. (A) For moderate salt stress-induced autophagy, excised leaves from 14-day-old rice seedlings were incubated in the Hoagland’s solution plus 150 mM NaCl, or 100 μM E-64d, or both NaCl and E-64d for 1 h. (B) For severe salt stress-induced autophagy, leaves were incubated in 250 mM NaCl and 100 μM E-64d for 1 h. (C) For TOR inhibition-induced autophagy, leaves were incubated in 2 μM AZD8055 and 1 μM Concanamycin A for 6 h. (D) For BTH-induced autophagy, 200 μM BTH and 100 μM E-64d for 6 h were used. (E) For ER stress-induced autophagy, 2 mM DTT and 100 μM E-64d for 4 h were used. At least three biological replicates were done for each treatment, and one representative replicate is shown. (F) A diagram illustrating the GFP cleavage assay and the chemicals used.

Figure 4

mRNA and protein levels of GFP and GFP-OsATG8a in rice transgenic lines. (A) Expression of GFP (27 kD) and GFP-OsATG8a (40 kD, one or two bands) were detected by immunoblotting and quantified by ImageJ. The protein level of GFP was normalized to Pro35S:GFP—L1 (set as 1), and the level of GFP-OsATG8a was normalized to Pro35S:GFP-OsATG8a—L1 (set as 1). Three T2 transgenic lines for each construct were shown. Coomassie Blue staining of RuBisCO large subunit was used as loading control. Three biological replicates were done, and one representative replicate is shown. (B) Schematic representation of the primers designed for qRT-PCR in (C–E). (C–E) qRT-PCR analysis of transgenic seedlings. Transcript levels of (C) GFP, (D) OsATG8a, and (E) OsATG8a-UTR were profiled and normalized to the Pro35S:GFP—L1 (C) or the wild-type control (D,E). Data are means ± SD (n = 4), one-way ANOVA followed by Tukey B test; p = 0.05. Different letters denote significant differences. Three biological replicates, each consisting of four technical repeats, were done, and one representative replicate is shown.

Figure 5

Identification of new ATG8-interacting proteins with immunoprecipitation-mass spectrometry analyses on GFP-OsATG8a marker lines. (A) Venn diagram showing the overlap in the number of proteins identified from three GFP-OsATG8a lines and the GFP control line, under normal growth condition (Mock). (B) Venn diagram showing the overlap in the number of proteins identified from three GFP-OsATG8a lines and the GFP control line, treated with 150 mM NaCl for 1 h (NaCl). (C) Gene Ontology (GO)-Biological Process (BP) enriched in all OsATG8a-interacting proteins (166 in total) identified from the three transgenic lines under normal growth condition (Mock). (D) GO-BP enriched in all 170 OsATG8a-interacting proteins identified after salt stress (NaCl). (E) Selected OsATG8a-interacting proteins, including regulators of autophagy and other trafficking routes, plasma membrane and tonoplast transporters, protein kinases and phosphatases, and other regulators of stress responses.