A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component (2022)
Figure 1

Figure 1: Xanthomonas blocks autophagy to enhance its pathogenicity

(A) RFP-ATG8g-labeled autophagosomes were quantified from plants syringe infected with mock or Xcv ΔxopQ (oD600=0.2) at 2 dpi in the presence or absence of ConA (bars = 20 μm). Puncta were calculated from z-stacks (15) of n=6 individuals using ImageJ. Central band of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Different letters indicate statistically different groups (P < 0.05) as determined by one-way ANOVA. The experiment was repeated twice with similar results.

(B) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ or mock infected N. benthamiana plants at 1 and 2dpi. Agrobacterium-mediated transient expression of AIMp-RFP serves as a control for autophagy suppression. Ponceau Staining (PS) served as a loading control. The experiment was repeated three times with similar results.

(C) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. Xcv ΔxopQ was co-infiltrated with Agrobacteria containing the luciferase reporter constructs. Coexpression of RFP-AIMp serves as a control for autophagy inhibition. Expression of the latter was confirmed with western blot (inset). Renilla (RLUC) and Firefly (FLUC) luciferase activities were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (***P<0.001) was revealed by Student's t-test. The experiment was repeated more than 3 times with similar results.

(D) RT-qPCR analysis of NbATG8-1.1/1.2, NbATG8-2.1/2.2 and NbJoka2 transcript levels upon challenge of N. benthamiana plants with Xcv ΔxopQ for 1 and 2 dpi compared to mock infected plants. Values represent expression relative to mock control of respective time point and were normalized to actin. Values are biological replicates (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (*P < 0.05, **P < 0.01, *** P < 0.001) was revealed by Student's t-test.

(E) Bacterial density in leaves of N. benthamiana infected with Xcv in the presence or absence autophagy suppressor AIMp-RFP. Leaves were syringe-infiltrated with OD600 = 0.0004, and colony-forming units were counted at 6 dpi. Compared to empty vector control (EV), AIMp expressing plants (n=6) harbour significantly more bacteria. Bacterial growth was repeated with the same result in 12 plants over two independent experiments. Red and yellow data points indicate independent repeats of the experiment. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (***P < 0.001) was revealed by Student's t-test. Expression of RFP-AIMp was verified at 6 dpi with an anti-RFP blot (inset).