Recruitment of the host plant heat shock protein 70 by Tomato yellow leaf curl virus coat protein is required for virus infection

Abstract

A functional capsid protein (CP) is essential for host plant infection and insect transmission of Tomato yellow leaf curl virus (TYLCV) and other monopartite begomoviruses. We have previously shown that TYLCV CP specifically interacts with the heat shock protein 70 (HSP70) of the virus insect vector, Bemisia tabaci. Here we demonstrate that during the development of tomato plant infection with TYLCV, a significant amount of HSP70 shifts from a soluble form into insoluble aggregates. CP and HSP70 co-localize in these aggregates, first in the cytoplasm, then in the nucleus of cells associated with the vascular system. CP-HSP70 interaction was demonstrated by co-immunopreciptation in cytoplasmic - but not in nuclear extracts from leaf and stem. Inhibition of HSP70 expression by quercetin caused a decrease in the amount of nuclear CP aggregates and a re-localization of a GFP-CP fusion protein from the nucleus to the cytoplasm. HSP70 inactivation resulted in a decrease of TYLCV DNA levels, demonstrating the role of HSP70 in TYLCV multiplication in planta. The current study reveals for the first time the involvement of plant HSP70 in TYLCV CP intracellular movement. As described earlier, nuclear aggregates contained TYLCV DNA-CP complexes and infectious virions. Showing that HSP70 localizes in these large nuclear aggregates infers that these structures operate as nuclear virus factories.

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.