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. 2018 Nov;98(4-5):363-373.
doi: 10.1007/s11103-018-0783-0. Epub 2018 Nov 3.

Citrus tristeza virus co-opts glyceraldehyde 3-phosphate dehydrogenase for its infectious cycle by interacting with the viral-encoded protein p23

Susana Ruiz-Ruiz  1 , Roberta Spanò  1   2 , Luis Navarro  3 , Pedro Moreno  3 , Leandro Peña  1 , Ricardo Flores  4
Affiliations

Citrus tristeza virus co-opts glyceraldehyde 3-phosphate dehydrogenase for its infectious cycle by interacting with the viral-encoded protein p23

Susana Ruiz-Ruiz et al. Plant Mol Biol. 2018 Nov.

Abstract

Citrus tristeza virus encodes a unique protein, p23, with multiple functional roles that include co-option of the cytoplasmic glyceraldehyde 3-phosphate dehydrogenase to facilitate the viral infectious cycle. The genome of citrus tristeza virus (CTV), genus Closterovirus family Closteroviridae, is a single-stranded (+) RNA potentially encoding at least 17 proteins. One (p23), an RNA-binding protein of 209 amino acids with a putative Zn-finger and some basic motifs, displays singular features: (i) it has no homologues in other closteroviruses, (ii) it accumulates mainly in the nucleolus and Cajal bodies, and in plasmodesmata, and (iii) it mediates asymmetric accumulation of CTV RNA strands, intracellular suppression of RNA silencing, induction of some CTV syndromes and enhancement of systemic infection when expressed as a transgene ectopically or in phloem-associated cells in several Citrus spp. Here, a yeast two-hybrid screening of an expression library of Nicotiana benthamiana (a symptomatic experimental host for CTV), identified a transducin/WD40 domain protein and the cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as potential host interactors with p23. Bimolecular fluorescence complementation corroborated the p23-GAPDH interaction in planta and showed that p23 interacts with itself in the nucleolus, Cajal bodies and plasmodesmata, and with GAPDH in the cytoplasm (forming aggregates) and in plasmodesmata. The latter interaction was preserved in a p23 deletion mutant affecting the C-terminal domain, but not in two others affecting the Zn-finger and one internal basic motif. Virus-induced gene silencing of GAPDH mRNA resulted in a decrease of CTV titer as revealed by real-time RT-quantitative PCR and RNA gel-blot hybridization. Thus, like other viruses, CTV seems to co-opt GAPDH, via interaction with p23, to facilitate its infectious cycle.

Keywords: Citrus tristeza virus; Closteroviruses; Plant RNA viruses; Virus-host interactions.

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Figures

Fig. 1
Fig. 1
Identification by yeast two hybrid screening of two proteins from a N. benthamiana expression library interacting with CTV-p23. Solid growth tests were performed on -His [± 3AT] plates. For each interaction, two independent clones and several dilutions of the diploid yeast cells culture normalized at 5 × 104 cells and expressing both bait and prey constructs were spotted on several selective media. a The DO-2 selective medium lacking tryptophan and leucine was used as a growth control, and the different dilutions were also spotted on the selective medium DO-3 lacking tryptophan, leucine and histidine. b Four different concentrations of 3-AT, an inhibitor of the product from gene HIS3, were added to the DO-3 plates to increase stringency and reduce possible auto-activation by p23. Empty vectors are denoted by ø, C+ refers to interaction positive control from Hybrigenics and C− to negative controls as indicated in the figure
Fig. 2
Fig. 2
Bimolecular fluorescence complementation assay in planta. Confocal laser-scanning microscopy of N. benthamiana leaves co-infiltrated with sYFPC-p23/sYFPN-p23 (a), sYFPC-GAPDH/sYFPN-GAPDH (b), and sYFPC-GAPDH/sYFPN-p23 (c). No fluorescence signal was observed in all pairwise p23 and GAPDH combinations with the binary vector expresing sYFPN or sYFPC alone (d). Overlays confirms the self-interaction of p23 in the nucleolus (No) and Cajal bodies (CB), and in plasmodesmata (PD), the self-interaction of GAPDH in the cytoplasm, and the interaction between p23 and GAPDH in the cytoplasm and plasmodesmata
Fig. 3
Fig. 3
Bimolecular fluorescence complementation assay in planta. Confocal laser-scanning microscopy of N. benthamiana leaves co-infiltrated with plasmids expressing GAPDH and three deletion and two alanine substitution mutants of p23. No fluorescence signal was observed in the negative controls
Fig. 4
Fig. 4
VIGS of GAPDH expression in the absence and presence of CTV. a Accumulation of GAPDH mRNA and GAPDH-small interfering RNAs (siRNAs) at 15 dpi in upper leaves of N. benthamiana plants that were co-infiltrated with the empty plasmids TRVRNA2 and TRVRNA1, and with plasmids TRVRNA1 and TRVRNA2-GAPDHC1 or TRVRNA2-GAPDHC2, in which two regions of the GAPDH-C cDNA from N. benthamiana were cloned separately. b At this stage (15 dpi), new leaves from these plants were agroinfiltrated with a plasmid for expressing a full-length cDNA of CTV-T36 and the accumulation of GAPDH mRNA and was examined 21 days later. Gel-blot hybridization was performed with a specific riboprobe following electrophoresis in denaturing agarose (0.8%) and polyacrylamide (17%) gels for detecting the GAPDH mRNA and siRNAs, respectively. The 5S RNAs stained with ethidium bromide served as a loading control
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