A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component (2022)
Figure 5

Figure 5: XopL is ubiquitinated in planta and degraded by NBR1-mediated selective autophagy

(A) GFP-XopL was coexpressed with GFP or AIMp-RFP. Proteins were separated by SDS-PAGE and detected by immunoblotting using the indicated antibodies. GFP blot was split for visualization purpose. Ponceau Staining (PS) served as a loading control. The experiment was repeated three times with similar results.

(B) Colocalization of RFP-XopL with GFP-Joka2 in N. benthamiana leaves. Imaging was performed 2 d after transient expression and images represent single confocal planes from abaxial epidermal cells (bars = 20 μm). The experiment was repeated twice with similar results.

(C) FRET FLIM measurements of GFP-Joka2 and RFP-XopL in N. benthamiana leaves. The freeRFP construct served as a negative control and RFP-ATG8E (n = 9) as a positive control. Scattered points show individual data points, color indicates biological repeats. The lifetime (in ns) of GFP-Joka2 (donor, n = 41) was significantly reduced in the presence of RFP-XopL (n = 40) but not in the presence of freeRFP (n = 35). 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. Significant differences were calculated using Wilcoxon rank sum test, with significantly different groups denoted by different letters. The experiment was repeated three times with similar results.

(D) Immunoprecipitation (IP) of GFP-XopL reveals association with NBR1. Immunoblots of input and IP samples from N. benthamiana plants transiently expressing GFP or GFP-XopL were probed with anti-GFP and anti-NBR1 antibodies. GFP blot was split for visualization purpose.

(E) Immunoprecipitation (IP) of GFP-XopL and GFP-XopL ΔE3 reveals association with NBR1. Immunoblots of input and IP samples from N. benthamiana plants transiently expressing GFP, GFP-XopL and GFP-XopL ΔE3 were probed with anti-GFP and anti-NBR1 antibodies. GFP blot was split for visualization purpose.

(F) GFP-XopL was transiently expressed in pTRV2, pTRV2-Joka2 and N. benthamiana WT plants. Expression of binary constructs was verified with an anti-GFP antibody. Joka2 silencing was verified using an anti-NBR1 antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.

(G) Growth of Xcv ΔxopQ in N. benthamiana plants silenced for Joka2 (pTRV2-Joka2) compared to control plants (pTRV2). Leaves were dip-inoculated with a bacteria suspension at OD600 = 0.2. and bacteria were quantified at 3 and 6 dpi. Red, blue, and green data points represent repeats of the experiments. 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. Significant differences were calculated using Student's t-test and are indicated by: **, P < 0.01. The experiment was repeated three times with similar trends.

(H) GFP-XopL, GFP-XopL ΔE3 were transiently expressed in N. benthamiana. RFP-AIMp was co-infiltrated to stabilize both XopL variants. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblot analysis of the precipitates using either anti-GFP or anti-ubiquitin antibodies. GFP served as a negative control. RFP-AIMp expression was verified by an anti-RFP antibody. GFP blot was split for visualization purpose. Asterisk indicates the GFP-XopL full-length protein. The experiment was repeated three times with similar results.

(I) GFP-XopL was transiently expressed in N. benthamiana. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation (IP) with the ubiquitin pan selector, followed by immunoblot analysis of the precipitates using either anti-GFP or anti-ubiquitin antibodies. GFP served as a control. Asterisk indicates the GFP-XopL full-length protein. GFP blot was split for visualization purpose. The experiment was repeated two times with similar results.

(J) Immunoblot analysis GFP-XopL and GFP-XopLK191A at 1 and 2 dpi using an anti-GFP antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated three times with similar results.

(K) HA-XopL and GFP-Joka2 and its variants were transiently expressed in N. benthamiana. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblot analysis of the precipitates using either anti-GFP, anti-ubiquitin and anti-NBR1 antibodies. GFP served as a control.