A plant virus movement protein regulates the Gcn2p kinase in budding yeast

Abstract

Virus life cycle heavily depends on their ability to command the host machinery in order to translate their genomes. Animal viruses have been shown to interfere with host translation machinery by expressing viral proteins that either maintain or inhibit eIF2α function by phosphorylation. However, this interference mechanism has not been described for any plant virus yet. Prunnus necrotic ringspot virus (PNRSV) is a serious pathogen of cultivated stone fruit trees. The movement protein (MP) of PNRSV is necessary for the cell-to-cell movement of the virus. By using a yeast-based approach we have found that over-expression of the PNRSV MP caused a severe growth defect in yeast cells. cDNA microarrays analysis carried out to characterise at the molecular level the growth interference phenotype reported the induction of genes related to amino acid deprivation suggesting that expression of MP activates the GCN pathway in yeast cells. Accordingly, PNRSV MP triggered activation of the Gcn2p kinase, as judged by increased eIF2α phosphorylation. Activation of Gcn2p by MP expression required a functional Tor1p kinase, since rapamycin treatment alleviated the yeast cell growth defect and blocked eIF2α phosphorylation triggered by MP expression. Overall, these findings uncover a previously uncharacterised function for PNRSV MP viral protein, and point out at Tor1p and Gcn2p kinases as candidate susceptibility factors for plant viral infections.

Figure 1. Expression of viral MPs of the 30 K superfamily interferes with yeast cell growth.

Growth kinetics of WT yeast cells cultured in SD medium +/- Doxycycline (DOX) and transformed with empty vector (pCM) (A), a full-length MPpnrsv expressing plasmid (B) or a truncated MPpnrsvΔHR expressing plasmid (C). (D) Quantitation of cell growth in SD medium+/- DOX of yeast cultures transformed with vector alone or with expressing MPpnrsv and MPpnrsvΔH plasmids. (E) Quantitation of cell growth in SD medium+/- DOX, of WT yeasts transformed with either pCM (lane 1) or with plasmids expressing PNRSV, BMV, CMV, TMV and GLFV MPs (lanes 2, 3, 4, 5 and 6, respectively). Data in D and E, represent the average +/- standard error (s.e.) of at least three independent experiments, each one done in triplicate (p<0.001). (F) Immunodetection of MPpnrsv (lane 3) and MPpnrsvΔHR (lane 1) in protein extracts from yeast cells transformed with the corresponding expression vectors or pCM as negative control (lane 2). (G) The same as in (F), but in protein extracts from yeast cells transformed with pCM (lane 1) or plasmids expressing PNRSV, BMV, CMV, TMV or GLFV MPs (lanes 2, 3, 4, 5 and 6, respectively) Coomassie Blue (CB) stained gel used as loading control).

Figure 2. MPpnrsv expression activates the GCN pathway in a TOR pathway-dependent manner.

(A) Immunodetection of phospho- eIF-2α levels in protein extracts from yeast cells transformed with pCM (lane 1), MPpnrsvΔHR (lane 2) and MPpnrsv (lane 3) expressing plasmids. Yeast cells were growth for 3 hours in SD medium without DOX. Even loading of gels was confirmed by CB staining of membranes after transfer. (B) Disruption of the GCN pathway alleviates the growth defect caused by MPpnrsv expression in yeast. Quantitation of cell growth in SD medium+/- DOX, of WT, gcn2Δ and gcn4Δ yeast strains expressing the MPpnrsv. (C) Rapamycin treatment alleviates the growth defect caused by MPpnrsv expression in yeast. Yeast cells expressing MPpnrsv were grown in SD medium +/- DOX (SD), the same medium with 200 µg/ml cycloheximide (C200), and with 100 µg/ml (R100) or 200 µg/ml (R200) Rapamycin respectively. Data in B and C represent the average +/- s.e. of at least three independent experiments, each one done in triplicate. (p<0.005) (D) Rapamycin treatment blocks phosphorylation of yeast eIF-2α triggered by MPpnrsv expression. Immunodetection of phospho- eIF-2α levels in protein extracts from yeast cells transformed with pCM and MPpnrsv expressing plasmids, grown in SD medium with (+R) or without (-R) 200 µg/ml Rapamycin for three hours. Coomassie Blue (CB) stained gel used as loading control.