ACT | Actin (polyclonal)

(A–D) Western blot analyses of S. lycopersicum midrib extracts. Three healthy (lanes 1–3) and three infected (lanes 4–6) plants were analyzed. Immunoreactive signals corresponding to actin and ER luminal binding protein (BiP) displayed the expected molecular weights of ?40 and ?78 kDa, respectively (A). Amount of loaded protein were checked by Ponceau-S staining (B). Quantification of immunoreactive luminescence signals was achieved by densitometric scanning of the respective actin (C) and BiP (D) bands. The mean of six replicates (±SD) was calculated for each sample. Actin T-value: –9.977; BiP T-value: 15.068. * denotes significant difference of the means (P-values ? 0.001).

Representative dephosphorylation profiles of phototropin1 after blue light exposure (120 µmol m?2 s?1 and 1h) in Arabidopsis wild-type and mutant (phot2 and rcn1) leaves. Dark, a dark-adapted control; 0, a sample collected just after illumination. The duration of the incubation in the darkness after the end of the illumination is indicated in minutes. Phosphorylation leads to the shift of the phototropin band towards higher mass. Samples treated with alkaline phosphatase are shown on the right. Anti-actin blots are presented as the loading reference. The results represent two out of 4–5 independent biological replicates.

Csp22 from Ralstonia solanacearum triggers additional early and late PRR?dependent immune responses in Solanaceae plants. (a) MAPK activation assay in leaves of 3? or 5?week?old Nicotiana benthamiana plants after treatment with 1 ?m csp22Rsol or water (mock) for 15 or 30 min as indicated. Immunoblots were analysed using antiphosphorylated MAPK antibody (??pMAPK). Immunoblots were also analysed using ??actin antibody to verify protein accumulation. Molecular weight (kDa) marker bands are indicated for reference. (b) Growth inhibition of N. benthamiana seedlings treated with 100 nm flg22Pto, csp22Rsol or water (mock) for 9 days. Values represent seedling weigh shown as percentage of mock?treated seedlings (average ± SE; n = 12). (c) Photograph of seedlings after exposure to the different elicitors in (b). (d) Growth of R. solanacearum GMI1000 in tomato (cultivar M82) leaves pretreated with 1 ?m csp22Rsol or water (mock) for 24 h and then syringe?infiltrated with a 106 cfu/mL bacterial inoculum. Bacterial growth was determined 3 days after inoculation. Values are mean ± SE (n = 3). Asterisks indicate significant differences compared to the mock control according to a Student's t?test (P < 0.001). All experiments were performed three times with similar results.

Constitutive overexpression of ATG5 or ATG7 in Arabidopsis stimulates lipidation of Atg8. (A) A schematic representation of two autophagy-related ubiquitin-like conjugation systems (Nakatogawa, 2013). Atg7 acts as an E1-like ligase by stimulating conjugation of two substrate pairs: of a ubiquitin-like protein Atg12 with its E2-like ligase, Atg10, and of a ubiquitin-like protein Atg8 with its E2-like ligase, Atg3. Atg12 is further transferred from Atg10 onto Atg5 protein in an E3-ligase-independent manner. The Atg12–Atg5 conjugate forms a complex with Atg16 and gains an E3-like ligase activity. Atg12–Atg5–Atg16 E3-like ligase stimulates conjugation of Atg8 with a phosphatidylethanolamine (PE), followed by anchoring of Atg8–PE on a membrane of a growing phagophore. Double green and double red asterisks indicate Atg12–Atg5 and Atg8–PE conjugates, respectively, also shown on the western blots on (B) and (D). (B) Atg5, but not Atg7, is a limiting component in the Atg12–Atg5 conjugation pathway. Western blot detection of Atg5 in rosette leaves of Col-0 (WT), two individual ATG5- or ATG7-overexpressing lines (ATG5 OE or ATG7 OE, respectively), and ATG5- or ATG7-knockout (atg5 or atg7, respectively) mutants. *Atg5; **Atg12–Atg5 conjugate. The experiment was repeated four times, using three individual lines of each overexpressor background. Western blot detection of actin and Ponceau staining were used as loading controls. (C) Densitometry of the Atg5–Atg12 conjugate in (B). Integrated density of bands corresponding to the Atg5–Atg12 conjugate were first normalized to the corresponding values for actin and then expressed as a percentage of the obtained value for the WT. Data represent means ±SE; n=6. Mean values denoted by the same letter do not differ significantly at P<0.001 (Student’s t-test). (D) Up-regulation of either ATG5 or ATG7 stimulates lipidation of Atg8. Seven-day-old seedlings of Col-0 (WT), atg5, ATG5 OE, atg7, and ATG7 OE genotypes were incubated for 3 d under 150 µE m–2 s–1 light, 16 h photoperiod (Light) or in the darkness (Dark). Total proteins were fractionated by ultracentrifugation and used for western blot analysis to detect free (*) and lipidated (**) forms of Atg8. C, crude extract; S, supernatant of 100000 g fraction; M, pellet of 100000 g fraction, containing membranes. (E) Densitometry of the Atg8–PE conjugate in (D). Integrated density of bands corresponding to the Atg8–PE in the crude fraction was expressed as a percentage of the integrated density of the total amount of Atg8 in the corresponding sample. Data represent means ±SE; n=6. Mean values denoted by the same letter do not differ significantly at P<0.001 (Student’s t-test). L, light; D, dark.

Constitutive overexpression of ATG5 or ATG7 stimulates formation of autophagosomes and increases autophagic flux. (A) Representative confocal microscopy images of the epidermal root cells expressing GFP–Atg8a in WT, ATG-knockout, and ATG-overexpressing backgrounds. Seedlings were grown under 150 µE m–2 s–1 light, 16 h photoperiod for 6 d and subjected to 0.5 µM ConA treatment for 16 h prior to imaging. Scale bars=10 µm. (B) The box-plot diagram shows the number of the GFP-positive puncta in the epidermal root cells in (A). The average number of puncta for two or three cells belonging to the same root was considered as a single measurement. Four to seven roots were imaged per genotype. The means of each genotype were compared with the WT using Dunnett’s test, assuming a Poisson distribution of the data. ***P<0.001; ?, outliers. (C) Increase of autophagic flux in ATG5- or ATG7-overexpressing plants confirmed by a higher rate of NBR1 degradation. Plants of the indicated genotypes were grown under 150 µE m–2 s–1. Rosette leaves were sampled at the onset of flowering (0 days after flowering, DAF) and 10 d later (10 DAF). Total protein extracts from sampled leaves were used for western blot analysis to detect NBR1 and actin. (D) Densitometry of the NBR1 signal in (C) and expression level of NBR1 in the same samples. Integrated density values for NBR1 were first normalized to actin and then expressed as a percentage of 0 DAF for the corresponding sample. Data represent means ±SE; n=6. The means of each genotype were compared with the WT using Dunnett’s test, ***P<0.001. Detection of the NBR1 transcript level in ATG-overexpressing plants confirmed that the decrease in NBR1 protein was caused by protein degradation and did not originate from transcriptional variation. qRT-PCR was performed on the same leaf material as in (C). Data represent means ±SE for each genotype, normalized to two reference genes (PP2A and HEL) and to 0 DAF; n=6. The means of each genotype were compared with the WT using Dunnett’s test, ***P<0.001. (E) Increased autophagic flux in ATG5- or ATG7-overexpressing plants additionally confirmed by detection of free GFP accumulation in the seedlings expressing GFP–Atg8a in the indicated backgrounds. Seven-day-old seedlings grown under normal conditions (150 µE m–2 s–1 light, 16 h photoperiod) were either transferred onto sucrose-depleted MS medium and kept in the darkness (–) or left growing under normal conditions (+). Total protein extracts from the whole seedlings of each genotype were used for western blot analysis with anti-GFP. The GFP–Atg8a fusion has a predicted molecular weight of ~40 kDa; free GFP has a predicted molecular weight of 27 kDa. To avoid quantification of saturated pixels, several exposures were used for different samples. Comparisons of the absolute integrated density values for each line were made using the same exposure. For more information, see the Materials and methods. (F) Densitometry of the GFP–ATG8a cleavage assay in (E) confirms elevated autophagic flux in ATG OE backgrounds. The experiment was repeated twice; at least three western blot assays were performed for each experiment. The figure shows a representative example. Free GFP was expressed as a percentage of the total amount of GFP for each sample in (E). Data represent means ±SE of three individual measurements. The means of each genotype were compared with the WT using Dunnett’s test, ***P<0.001.