Figure 3(A) Interaction of XopL with SH3P2 in yeast two-hybrid assays. XopL fused to the GAL4 DNA-binding domain was expressed in combination with SH3P2 fused to the GAL4 activation domain (AD) in yeast strain Y190. Cells were grown on selective media before a LacZ filter assay was performed. pSV40/p53 served as positive control, while the empty AD or BD vector served as negative control. NtSH3P2 = Nicotiana tabacum SH3P2. -LT = yeast growth on medium without Leu and Trp, -HLT = yeast growth on medium lacking His, Leu, and Trp, indicating expression of the HIS3 reporter gene. LacZ, activity of the lacZ reporter gene.(B) Coimmunoprecipitation of GFP-XopL with AtSH3P2-HA. GFP-XopL or GFP were transiently coexpressed with AtSH3P2-HA in leaves of N. benthamiana. After 48 h, total proteins (Input) were subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblot analysis using either anti-GFP or anti-HA antibodies. GFP blot was split for visualization purpose. AtSH3P2 = Arabidopsis thaliana SH3P2. Two repetitions with similar results have been conducted.(C) Visualization of protein interactions in planta by the bimolecular fluorescence complementation assay. Yellow fluorescent protein (YFP) confocal microscopy images show Nicotiana benthamiana leaf epidermal cells transiently expressing VenusN173-XopL in combination with AtSH3P2-VenusC155. A positive control showing the dimerization of fructose-1,6-bisphosphatase (FBPase) within the cytosol. White arrows indicate reconstitution of YFP fluorescence. The red structures indicate autofluorescence of chloroplasts. The combination of VenusN173-XopL with FBPase-VenusC155 or VenusN173-FBPase with AtSH3P2-VenusC155 do not induce YFP fluorescence and serve as negative controls. Bars = 20 μm.(D) Colocalization analysis of GFP-XopL with SH3P2-RFP, RFP-ATG8e and RFP-ATG8g in N. benthamiana leaves. Imaging was performed 2 d after transient expression and images represent single confocal planes from abaxial epidermal cells (scale bars = 20 μm and 10 μm, lower panel). White arrows indicate colocalization of GFP and RFP signals. The experiment was repeated twice with similar results.(E) Total proteins were extracted 48 hpi with A. tumefaciens harboring the respective GFP-XopL, HA-XopL and SH3P2-GFP expression constructs. SH3P2-GFP protein levels (lower band) were detected using an anti-GFP antibody. Expression of the XopL was verified using an anti-HA or anti-GFP antibody. Expression of GUS-HA served as a control. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.(F) Growth of Xcv and Xcv ΔxopL strains in roq1 N. benthamiana plants silenced for SH3P2 (pTRV2-SH3P2) 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. Experimental repeats are indicated by data points in red (n=8) and yellow (n=6) data points. 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. Different letters indicate statistically significant differences (P < 0.05) as determined by one-way ANOVA.