Figure 1.
TYLCV CP and cellular HSP70 in total protein extracts of tomato leaf and stem during the progress of TYLCV infection.
CP (upper panel) and HSP70 (middle panel) were western blot immuno-detected at 14, 21, 28, 35, 42 and 56 days after the onset of whitefly-mediated inoculation (dpi). Coomassie blue stain of total proteins (lower panel). Molecular weight markers a–g (in kDa): (a) 17, (b) 26, (c) 34, (d) 43, (e) 55, (f) 72, and (g) 95.
Figure 2.
Viral CP and plant HSP70 in leaf and stem soluble and insoluble protein fractions.
Western blot analysis of the leaf and stem homogenates from not infected and TYLCV-infected tomatoes were fractionated into insoluble debris and cell wall (P), 3000 g pellet (P3) and soluble protein (S3). Coomassie blue stain of total proteins (lower panel). Molecular weight markers a–g (in kDa): (a) 17, (b) 26, (c) 34, (d) 43, (e) 55, (f) 72, and (g) 95.
Figure 3.
Distribution of viral CP and plant HSP70 following sedimentation on 10–50% sucrose gradients of native proteins extracted from tomato leaf and stem.
Leaf and steam homogenates were prepared from tomato plants before (A) and at 28 days after the onset of TYLCV infection (B). Gradients were divided into 10 fractions, 1 (top) to 10 (bottom) and concentrated about 20 times by TCA precipitation (see Materials and Methods). Aliquots were subjected to SDS-PAGE. The gels were stained with Coomassie blue (total protein) and Western blotted using anti-CP and anti-HSP70 antibodies. Anti-CP recognized an additional minor 31 kDa polypeptide. Molecular weight markers (noted as m) a–g (in kDa): (a) 17, (b) 26, (c) 34, (d) 43, (e) 55, (f) 72, and (g) 95.
Figure 4.
Co-immunoprecipitations of plant HSP70 by viral CP and vice versa in infected tomato tissues.
Figure 5.
Co-localization of CP and HSP70 in cytoplasm and nucleus of infected tomato leaf and stem at 28 and 49 dpi.
Leaf: cross-section through the midrib (P, phloem; LB, leaf blade). Stem: cross section between true leaves 2 and 3 (IP and EP, internal and external phloem; PI, pith). Fluorescent microscopy using primary anti-CP antisera and Cy3-labeled secondary antibody, primary anti-HSP70 antisera and Cy2-labeled secondary antibody; nuclei were DAPI stained. L4 and L4n: leaves 4 weeks after the onset of infection, without and with nuclei stain, respectively; L7 and L7n: same but 7 weeks after the onset of infection. S4 and S4n: stems 4 weeks after the onset of infection, without and with nuclei stain, respectively; S7 and S7n: same but 7 weeks after the onset of infection. Viral CP appears as red, cellular HSP70 as green, and nuclei as blue spots; CP in nuclei appears as violet, HSP70 in nuclei as light blue spots; CP co-localizing with HSP70 appears as orange to yellow spots (yellow arrows), CP co-localizing with HSP70 in nuclei - as pink to white spots (pink arrows). Bar: 50 µm, except for S4 and S4n, 100 µm.
Figure 6.
Changes affecting TYLCV and HSP70 in plants treated with quercetin.
(A) Western blot analysis of leaf cytoplasmic and nuclear proteins from 28 dpi tomatoes before and after quercetin treatment (400 µM for four days). lower panel: Coomassie blue stain of total proteins; molecular weight markers a–g (in kDa): (a) 17, (b) 26, (c) 34, (d) 43, (e) 55, (f) 72, and (g) 95. (B) Tomato leaflets at 28 dpi were incubated for 4 days with 400 µM quercetin, TYLCV DNA amounts were estimated by qPCR analysis. (C) N. benthamiana epidermal cells transiently expressing GFP-CP following infiltration with quercetin (800 µM) or DMSO (control). Bar: 100 µm.