pubmed.ncbi.nlm.nih.gov

Unravelling the involvement of cilevirus p32 protein in the viral transport - PubMed

  • ️Fri Jan 01 2021

Unravelling the involvement of cilevirus p32 protein in the viral transport

Mikhail Oliveira Leastro et al. Sci Rep. 2021.

Abstract

Citrus leprosis (CL) is a severe disease that affects citrus orchards mainly in Latin America. It is caused by Brevipalpus-transmitted viruses from genera Cilevirus and Dichorhavirus. Currently, no reports have explored the movement machinery for the cilevirus. Here, we have performed a detailed functional study of the p32 movement protein (MP) of two cileviruses. Citrus leprosis-associated viruses are not able to move systemically in neither their natural nor experimental host plants. However, here we show that cilevirus MPs are able to allow the cell-to-cell and long-distance transport of movement-defective alfalfa mosaic virus (AMV). Several features related with the viral transport were explored, including: (i) the ability of cilevirus MPs to facilitate virus movement on a nucleocapsid assembly independent-manner; (ii) the generation of tubular structures from transient expression in protoplast; (iii) the capability of the N- and C- terminus of MP to interact with the cognate capsid protein (p29) and; (iv) the role of the C-terminus of p32 in the cell-to-cell and long-distance transport, tubule formation and the MP-plasmodesmata co-localization. The MP was able to direct the p29 to the plasmodesmata, whereby the C-terminus of MP is independently responsible to recruit the p29 to the cell periphery. Furthermore, we report that MP possess the capacity to enter the nucleolus and to bind to a major nucleolar protein, the fibrillarin. Based on our findings, we provide a model for the role of the p32 in the intra- and intercellular viral spread.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

The cileviruses MPs complement the cell-to-cell and systemic movement of the AMV infectious clone. Analysis of the cell-to-cell and systemic transport of the hybrid AMV RNA 3 in which its MP gene was exchanged with the corresponding genes (MPs) of CiLV-C and CiLV-C2 and the genes p29, p15, p61 and p24 of CiLV-C. (A) Infection foci observed in P12 plants inoculated with RNA 3 transcripts from pGFP/A255/CP derivatives carrying the AMV MP lacking the C-terminal 44 residues (A44) (MP), the AMV MP wild type (MP:A44), and the aforementioned heterologous MPs alone (right panels; MP) or fused to A44 (left panels; MP:A44). The schematic representation shows the GFP/A255/CP AMV RNA 3, in which the open reading frames, represented by large boxes, correspond to the green fluorescent protein (GFP), the movement protein (MP) and the coat protein (CP). Short box corresponds the C-terminal 44 amino acids of the AMV MP, meanwhile arrows represent subgenomic promoters. The numbers after the viral acronym represent the total amino acids residues of the corresponding MP. The NcoI and NheI restriction sites used for insertions of the MPs are indicated, as well as the restriction sites BspHI, PciI and NheI for insertions of the other CiLV-C genes. White bars represent to 500 μm, 2 mm and 10 mm. N.D non-determined. (B) Histograms represent the average of the area in mm2 of 80 independent infection foci at 2 and 3 days post-inoculation (dpi). Error bars indicate the standard deviation. Student’s t-test and significance was set at p < 0.05. The p-values obtained from comparison between pairs of groups are presented. (C) Tissue-printing analysis of P12 plants inoculated with the AMV RNA 3 derivatives showed in A but lacking the 5′ proximal GFP gene. Plants were analyzed at 14 dpi by printing the transversal section of the corresponding petiole from inoculated (I) and upper (U) leaves. Mock corresponds to non-inoculated plant.

Figure 2
Figure 2

The cilevirus MP is able to transport infectious viral complexes. Analysis of cell-to-cell transport of the hybrid AMV RNA3, carrying different MP genes and the mutated coat protein gene (CP 199) defective in virus particles formation. The schematic representation corresponds to the same constructs indicated in Fig. 1A, in which the CP gene was replaced by the CP 199 gene from the construct pGFP/BMV:A44/CP199 by exchanging the NheI-PstI fragment. P12 plants were inoculated with transcripts derived from the AMV RNA 3 variants expressing AMV wt (positive control), CiLV-C, CiLV-C2 and TSWV (negative control) MPs. The images of foci formation or single-cell GFP foci were observed at 2 days post-inoculation (dpi) using a Leica Stereoscopic Microscope. Each infection foci image is representative of the inoculation of three leaves per plant and three plants inoculated for each chimeric AMV construct. White bars represent 0.5 mm to 2 mm.

Figure 3
Figure 3

The C-terminus of cileviruses MP is dispensable for cell-to-cell movement. (A) Alignment of amino acids sequence of the C-terminal region of CiLV-C and CiLV-C2 MPs. Residues common to the two sequences are shown in blue. Red dotted line indicates the most variable region at the C-termini, corresponding the last 36 residues of the MP. The alignment was performed using the software SnapGene 4.3.10. (B) Infection foci observed in P12 plants inoculated with RNA 3 transcripts carrying the C-terminal truncated MP genes of cileviruses. The schematic representation shows the chimeric GFP/A255/CP AMV RNA 3 represented in Fig. 1A and the C-terminal deletions of the MP proteins analyzed. Amino acid numbers 292 and 297 correspond to wild-type (wt) size of the corresponding MP proteins, meanwhile residues P, E, D, K, V, H, T and R for CiLV-C2 and V, G for CiLV-C correspond to the last amino acid included in the corresponding truncation. Superscript asterisks refer to the residues shown in alignment (A) correlated by their respective colors. Each infection foci image is representative of the inoculation of three leaves per plant and three plants inoculated for each chimeric AMV construct at 2 dpi. White bars correspond to 200 μm. (C) Histograms represent the average of the area in mm2 of 80 independent infection foci at 2 and 3 dpi of the P12 plants infected with hybrids AMV RNA 3 containing the wild type and truncated CiLV-C2 MPs P-Δ266–292, E-Δ225–292, D-Δ242–292 and K-Δ232–292 lacking -27, -38, -51 and -61 residues, respectively. Error bars indicate the standard deviation. Student’s t-test and significance was set at p < 0.05. The p-values obtained from comparison between pairs of groups are presented. (D) Tissue-printing analysis of P12 plants inoculated with a variant of AMV RNA 3 expressing the CiLV-C wt (297)(1), CiLV-C Δ228–297 (V)(2), CiLV-C2 wt (292)(3), CiLV-C2 Δ222–292 (T)(4) and AMV MP (AMV)(5). Plants were analyzed at 14 dpi by printing the transversal section of the corresponding petiole from inoculated (I) and upper (U) leaves.

Figure 4
Figure 4

The C-terminal of the cileviruses MP is intrinsically related to the tubule formation. Analysis of tubule formation from transient expression of the truncated MP proteins of the CiLV-C and CiLV-C2 fused at their C-termini with eGFP (green filled circle) on the surface of N. benthamiana protoplasts. Three infiltrated leaves per construct were used for protoplasts isolation. Protoplasts were purified after 1 day post-infiltration and the fluorescence GFP signal was captured 16 h post-protoplasts purification with a Zeiss LSM 780 confocal laser-scanning microscope. Each image-frame expressing GFP represents the visualization of several protoplasts (about 15 to 20) per assay for each MP construct analyzed. The green (GFP), transmitted light (TL) channels and merged images are shown. The scheme indicates the expression cassette introduced into the binary vector pMOG800 with the 35S cauliflower mosaic virus promotor region (black arrow box), the MP gene (gray box) carrying the eGFP report gene (green box) fused at their C-termini and the potato proteinase inhibitor II terminator region (PoPit) (blue arrow box). The number of deleted residues for each truncated MP is shown in blue. Short tubules are visualized for the MP truncated versions with maximum limit of deletions in movement functions still able to generate cell-to-cell spread (CiLV-C Δ228–297 and CiLV-C2 Δ222–292), while no tubules were observed for the constructions unable to generate movement (CiLV-C Δ225–297 and CiLV-C2 Δ218–292). The panels i–iii shown merged images of protoplasts highlighting the presence of short tubules (white arrows). T.L brightness was lowered to facilitate the visualization of short tubules. In panel ii, the dotted line simulates the cell periphery. Red bars correspond to 5–10 μm.

Figure 5
Figure 5

The C-terminal region of cileviruses MP is necessary for correct co-localization with plasmodesmata. Transient expression of CiLV-C wild type (a), CiLV-CΔ228–297 (b), CiLV-CΔ225–297 (c), CiLV-C2 wild type (d), CiLV-C2Δ222–292 (e) and CiLV-C2Δ218–292 (f) MPs, carrying the GFP (green filled circle) in N. benthamiana leaves. Fluorescence signal was captured at 72 h post-infiltration. The callose deposits were stained using aniline blue (blue filled circle). The blue and green arrows indicate the callose deposits in plasmodesma and MP, respectively; while the white arrows indicate co-localization between them. The blue (aniline blue), the green (GFP), transmitted light (TL) channels and merged images are shown in the figure. Images on the left show the tubule formation of N. benthamiana protoplast transiently expressing the correspondent MP construct. The MP punctate structures co-localize at the cell periphery with the fluorochrome for the MP wt constructs (a,d), while the truncated MP constructs show no co-localization or partial co-localization (b,c,e,f). In a higher magnification image, it is shown the complete (i,iv) or partial (ii,iii,v,vi) co-localization of the MP with callose deposits in the plasmodesmata, and the chart of fluorescence intensities further confirms the co-localization. Plot shows green and blue fluorescence intensities, indicated by MP:GFP and aniline blue, in the selected region of interest (red arrows). Distance measurement starts from the base to the tip of the arrows (x axis). The mean SD of Person Correlation Coefficient (PCC) is given in the merged image. PCC was measured using the Fiji co-localization plugin for three independent images from approximately 100 individual plasmodesmata. The dotted line in the transmitted light image indicates the cell wall (CW). Cyt cytoplasm. Red and white bars correspond to 5–10 and 20 μm, respectively.

Figure 6
Figure 6

Redistribution of the coat protein (p29) from the cytoplasm to callose deposits in plasmodesma by interaction with the MP of CiLV-C. (A) Subcellular localization of the CiLV-C p29 and MP fused with eGFP (green filled circle) transiently expressed in N. benthamiana leaves visualized at 72 h post-infiltration. White boxes correspond to high magnification to highlight callose deposits. White and red arrows indicate small punctate structures and large agglomerates of p29 (a). The green arrow in enlarged image in (b) indicates MP punctate structures along the cell membrane. (c,d) show the callose deposits stained with aniline blue (blue filled circle). White and blue arrows indicate regions of callose deposition at the cell membrane and the co-localization among the callose deposits in plasmodesma with the p29 or MP proteins, respectively. (B) Expression assay to determine the re-co-localization of the p29 when co-expressed with the MP. The p29-eGFP was co-expressed with the MP-HA (a). Red and white arrows indicate p29 large and small structure, respectively. The callose staining (b) shows the co-localization between the p29 with callose deposits in plasmodesmata (blue arrow). (C) Sub-cellular localization of the mutated MP CiLV-C MPΔ228–297 (a) lacking the C-terminus, and the remaining N-terminal region (CiLV-C MPΔ1–227) (b), also stained with aniline blue fluorochrome (c). (D) BiFC analysis to determine the MP region responsible for redirecting the coat protein. The C- and N-fragment of MP (a/c,b, respectively) fused at their C-terminus with CYFP were co-expressed with the cognate p29 protein fused at its C-terminus with the NYFP counterpart. Red and blue arrows indicate agglomerates dispersed throughout the cytoplasm and punctate structures along cell periphery, respectively. In panel c, the dark blue and green arrows indicate the callose deposits and MP, respectively; while the light blue arrow indicates co-localization between them. The mean SD of PCC is given in the merged image that shows callose staining. The BiFC fluorescence was visualized at four days pot-infiltration. The blue (aniline blue), the green (GFP), transmitted light (T.L) channels and merge images are shown in the figure. White and red bars correspond to 50 and 10 μm, respectively.

Figure 7
Figure 7

The cileviruses MP interacts with fibrillarin (Fib2) into the nucleolar compartment. (A) The CiLV-C MP (a) and CiLV-C2 MP (b) carrying the eGFP (green filled circle) were solely expressed (a,b) and co-expressed (c) with a nucleus marker (red filled circle) in leaf epidermal cells of N. benthamiana plants. The white arrows indicate the diffuse MP GFP signal into the nucleus (a,b) which co-localize with the nucleus marker. (c), histogram represents the nuclear GFP intensity observed with the CiLV-C and CiLV-C2 MPs. GFP intensity signals of 20 distinct nuclei for each construct were measured using Image J (version 2.0r) Macros plugin. The nucleus GFP signal is represented in percentage. Student’s t-test and significance was set at p < 0.05. The p-value obtained from comparison between pair of groups is presented. (B) BiFC analysis of the interaction between CiLV-C/CiLV-C2 MPs and Fib2. N. benthamiana fibrillarin was targeted at its C-terminus with NYFP and CYFP and co-expressed with the MP labelled at its N- or C-terminus with the NYFP or CYFP. Representative protein pair combinations are indicated at the left or bottom of each image. (a) Merged image indicating YFP fluorescence return in several nuclei. Magnification images shows YFP signal into the nucleus, suggesting interaction between MP of CiLV-C (b) or CiLV-C2 (c) and Fib2. Negative control (d) corresponds to the expression of the Fib2 protein in combination with N-Cyt vector and NoLS construct that correspond to a nucleolar peptide signal (RKRHAKKK) fused at the C-terminus of the YFP fragments. Positive control (e) corresponds to the dimerization of the Fib2 protein by Fib2-NYFP + Fib2-CYFP co-expression. The fluorescent signals were visualized at 72 h post-infiltration and, for the BiFC, at four days post-infiltration. The green (GFP), red (nucleus marker), transmitted light (T.L) channels and merged images are shown in the figure. Withe and red bars correspond to 50 and 10 μm, respectively. The BiFC images displayed are representative of at least three independent experiments. (C) Co-immunoprecipitation of CiLV-C2 MP with Fib2. Agrobacteria cultures containing MP-HA and Fib2-3xMyc plasmids, were co-infiltrated into N. benthamiana leaves and extracts were analyzed at 3 days post infiltration. HA and Myc antibodies were used in the western blots. A, leaf extract treated with a non-denaturing buffer; B, leaf extract treated with RIPA buffer; C+, positive controls (samples non-immunoprecipitated) and IP, immunoprecipitated samples. The + and − signs indicate the presence or absence of the corresponding proteins in the leaf extracts.

Figure 8
Figure 8

Model for the role of the cileviruses MP in viral intracellular and intercellular transport. The cileviruses replication occurs in the membranes of endoplasmic reticulum (ER) resulting in larger viroplasms. A portion of the MP is transported into the nucleolus of the cell to bind to fibrillarin. The possibility to form a complex between MP plus fibrillarin that could exit the nucleus and interacts with vRNA and/or CP (p29) to form infectious vRNPs, is an open question. The MP, a membrane spanning protein, may anchors the vRNP complex to the ER membrane network, which traffics by the ER system to neighboring cells, facilitating the passage through plasmodesmata by the tubule formation. A possible alternative route may be mediated by the capacity of p29 to interact with MP and to associate with actin, thus anchoring the infectious complexes along the microfilaments (MF), guiding the vRNPs throughout the cytoplasm to the cell periphery. The virus particle is not required for the intercellular transport for this MP (indicating its capability to transport viral complexes different to virus particles intra and intercellularly); furthermore, the MP can transport the infectious complex cell-to-cell and systemically independent of the CP assistance. The redirection of p29 by MP to the plasmodesma could also be implicated to initiate viral replication in the newly infected cells.

Similar articles

Cited by

References

    1. Morozov SY, Solovyev AG. Triple gene block: Modular design of a multifunctional machine for plant virus movement. J. Gen. Virol. 2003;84:1351–1366. doi: 10.1099/vir.0.18922-0. - DOI - PubMed
    1. Lazareva EA, et al. A novel block of plant virus movement genes. Mol. Plant Pathol. 2017;18:611–624. doi: 10.1111/mpp.12418. - DOI - PMC - PubMed
    1. Lucas WJ. Plant viral movement proteins: Agents for cell-to-cell trafficking of viral genomes. Virology. 2006;344:169–184. doi: 10.1016/j.virol.2005.09.026. - DOI - PubMed
    1. Navarro JA, Sanchez-Navarro JA, Pallas V. Key checkpoints in the movement of plant viruses through the host. Adv. Virus Res. 2019;104:1–64. doi: 10.1016/bs.aivir.2019.05.001. - DOI - PubMed
    1. Hofmann C, Niehl A, Sambade A, Steinmetz A, Heinlein M. Inhibition of tobacco mosaic virus movement by expression of an actin-binding protein. Plant Physiol. 2009;149:1810–1823. doi: 10.1104/pp.108.133827. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances