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. 2021 Mar 4;17(3):e1009370. doi: 10.1371/journal.ppat.1009370

Fig 5. Silencing NbMIP1s delays RSV infection, and expressing NbMIP1.4b enhances the ability of NSvc4 to rescue the cell-to-cell movement of PVX(Δp25)-eGFP.

Fig 5

(A) The phenotype of silencing NbMIP1s by TRV-based VIGS. The image was taken at 10 days after inoculation of TRV. (B and C) The symptom of TRV-NbMIP1 and TRV-GFP pre-inoculated plants at 12 dpi of RSV. Bars, 1 cm. (D) Time-course analysis of disease severity index (DSI). The experiments were carried out 3 times independently. Value at each point represents the mean of DSI ± SD. (E) Detection of RSV CP accumulation in TRV-NbMIP1 and TRV-GFP pre-inoculated plants. The leaves from RSV-infected plans in (C) were harvested. Total protein was extracted for western blotting. The protein level of RSV CP represented virus accumulation level, and the protein level of NbMIP1s indicated silencing efficiency. (F) Detection of relative protein level of NSvc4 in TRV-NbMIP1 and TRV-GFP pre-inoculated plants. The total protein of the leaves showed similar symptom severity were extracted for western blotting. The relative levels of NSvc4 to RSV CP were calculated and normalized by setting the value of the first lane of TRV-GFP plant as 1.0. (G and H) Measurements of the chlorotic lesion diameter on local leaves. Images of local leaves from TRV-NbMIP1 and TRV-GFP pre-inoculated plants were taken at 11 dpi. Areas of leaves were magnified to show the detail of chlorotic lesions. Bars, 1.0 cm. The diameter of these lesions was measured and analyzed by student’s t-test (two-sided, ***P < 0.001); the mean and the number of measured lesions (n) were labeled. (I-L) Analysis of the effects of NbMIP1.4b and HPD-motif-deleted mutant to the NSvc4 rescued cell-to-cell movement of PVX(Δp25)-eGFP. Agrobacterium carrying NbMIP1.4b-Myc, or NbMIP1.4b(ΔHPD)-Myc, or empty vector was mixed with agrobacteria carrying 800-fold-diluted PVX(Δp25)-eGFP, NSvc4, and TBSV p19. Each mixture was infiltrated into each half of the same leaf. The leaves were observed under fluorescence stereoscopic microscopy at 5 dpi. The area of fluorescence was measured by ZEN lite software and analyzed by student’s t-test (two-sided, *** P < 0.001, n.s., not significant); the mean and the number of measured area (n) were labeled. Meanwhile, leaves were harvested for western blotting to confirm the expression of NbMIP1.4b-Myc or its mutant.