Palmitoylation of γb protein directs a dynamic switch between Barley stripe mosaic virus replication and movement (2022)
Figure 7

Figure 7. Palmitoylation of γb facilitation of PM localization and BSMV cell-to-cell movement

A Plasma membrane (PM) targeting of γb in BSMVγb-GFP-infected N. benthamiana leaves plasmolized at 72 hpi. Infiltrated leaf sections were cut into small strips and soaked in 5% NaCl for 5 to10 min prior to confocal microscopy observations. The dotted lines in the merged panel highlight the cell wall (CW) after plasmolysis. GFP fluorescence was retracted along with the PM to reveal Hechtian strands connecting the PM to the CW (white arrow heads). Bars, 20 μm.

B Fractionation assay to detect γb membrane-associations. Separation of total protein (T) extracted from BSMV infected leaves into soluble (S) and membrane (M) fractions by ultracentrifugation at 100,000 g. PEPC and BiP, respectively, provide soluble and membrane fraction markers.

C Aqueous two-phase partitioning of γb PM localization. The microsomal pellet from BSMVγb-GFP-infected leaves was obtained by ultracentrifugation at 100,000 g, and the two-phase fractionation procedure was used to separate the plasma membrane (upper phase) from other cellular membranes (lower phase). H+-ATPase is a PM marker.

D PM associations of γb-GFP and γb3CS-GFP in N. benthamiana protoplasts. The first and second columns show localization of γb at 3 dpi after infiltrations for transient expression of γb-GFP and γb3CS-GFP. The third and fourth columns show γb localization in BSMVγb-GFP and BSMV3CS-GFP-infected leaves. PIP2A provided a PM marker (Nelson et al., 2007). Figures below the confocal images show the normalized fluorescence intensities of the GFP and mCherry proteins along the white dashed lines in the merged images. Scale bars, 10 μm.

E Western blot analysis of subcellular fractions of BSMV infected N. benthamiana leaves at 3 dpi. The S3 and S30 fractions are the 3000 and 30000 g supernatants, and P30 is the 30000 g pellet. Membrane associated γb was released by 1% Triton X-100 treatment.

F Western blot detection of BSMV accumulation in N. benthamiana plants. A. tumefaciens mixtures harboring RNAα and RNAβmTGB2, along with RNAγ, RNAγγb-ROP, or RNAγγb-mROP, were agroinfiltrated into N. benthamiana leaves. Western blotting to detect virus accumulation at 3 dpi used anti-TGB1 or anti-CP antibodies. Actin provided loading controls.

G Analysis of the effects of enhanced PM association of γb on cell-to-cell virus movement with the BSMV duplex fluorescence (dfBSMV) reporter system. Mixtures of A. tumefaciens containing dfBSMV, dfBSMVγb-GFP-ROP, and dfBSMVγb-GFP-mROP were infiltrated into N. benthamiana leaves. Infiltrated leaves were observed by confocal microscope at 3 dpi. Scale bars, 100 μm. The determination of the movement capacity of the viruses depicted in each panel as descripted in Fig 3B (n = 6).

Data information: In (A, D, and G), representative data are shown and three biological replicates had similar results. In (G), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).

Source data are available online for this figure.