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Nicotiana benthamiana is an important model plant for plant-microbe interaction studies. Here, we compared ribosome profiles and riboproteomes of healthy and infected N. benthamiana plants. We affinity purified ribosomes from transgenic leaves expressing a FLAG-tagged ribosomal large subunit protein RPL18B of Arabidopsis thaliana. Purifications were prepared from healthy plants and plants that had been infiltrated with Agrobacterium tumefaciens carrying infectious cDNA of Potato virus A (PVA) or firefly luciferase gene, referred to here as PVA- or Agrobacterium-infected plants, respectively. Plants encode a number of paralogous ribosomal proteins (r-proteins). The N. benthamiana riboproteome revealed approximately 6600 r-protein hits representing 424 distinct r-proteins that were members of 71 of the expected 81 r-protein families. Data are available via ProteomeXchange with identifier PXD011602. The data indicated that N. benthamiana ribosomes are heterogeneous in their r-protein composition. In PVA-infected plants, the number of identified r-protein paralogues was lower than in Agrobacterium-infected or healthy plants. A. tumefaciens proteins did not associate with ribosomes, whereas ribosomes from PVA-infected plants co-purified with viral cylindrical inclusion protein and helper component proteinase, reinforcing their possible role in protein synthesis during virus infection. In addition, viral NIa protease-VPg, RNA polymerase NIb and coat protein were occasionally detected. Infection did not affect the proportions of ribosomal subunits or the monosome to polysome ratio, suggesting that no overall alteration in translational activity took place on infection with these pathogens. The riboproteomic data of healthy and pathogen-infected N. benthamiana will be useful for studies on the specific use of r-protein paralogues to control translation in infected plants., (© 2018 The Authors. Molecular Plant Pathology published by BSPP and John Wiley & Sons Ltd.)

Potyviral helper component proteinase (HCPro) is a well-characterized suppressor of antiviral RNA silencing, but its mechanism of action is not yet fully understood. In this study, we used affinity purification coupled with mass spectrometry to identify binding partners of HCPro in potyvirus-infected plant cells. This approach led to identification of various HCPro interactors, including two key enzymes of the methionine cycle, S-adenosyl-L-methionine synthase and S-adenosyl-L-homocysteine hydrolase. This finding, together with the results of enzymatic activity and gene knockdown experiments, suggests a mechanism in which HCPro complexes containing viral and host proteins act to suppress antiviral RNA silencing through local disruption of the methionine cycle. Another group of HCPro interactors identified in this study comprised ribosomal proteins. Immunoaffinity purification of ribosomes demonstrated that HCPro is associated with ribosomes in virus-infected cells. Furthermore, we show that HCPro and ARGONAUTE1 (AGO1), the core component of the RNA-induced silencing complex (RISC), interact with each other and are both associated with ribosomes in planta. These results, together with the fact that AGO1 association with ribosomes is a hallmark of RISC-mediated translational repression, suggest a second mechanism of HCPro action, whereby ribosome-associated multiprotein complexes containing HCPro relieve viral RNA translational repression through interaction with AGO1., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

We report here that the acidic ribosomal protein P0 is a component of the membrane-associated Potato virus A (PVA) ribonucleoprotein complex. As a constituent of the ribosomal stalk, P0 functions in translation. Although the ribosomal stalk proteins P0, P1, P2, and P3 are all important for PVA infection, P0 appears to have a distinct role from those of the other stalk proteins in infection. Our results indicate that P0 also regulates viral RNA functions as an extraribosomal protein. We reported previously that PVA RNA can be targeted by VPg to a specific gene expression pathway that protects the viral RNA from degradation and facilitates its translation. Here, we show that P0 is essential for this activity of VPg, similar to eIF4E/eIF(iso)4E. We also demonstrate that VPg, P0, and eIF(iso)4E synergistically enhance viral translation. Interestingly, the positive effects of VPg and P0 on viral translation were negatively correlated with the cell-to-cell spread of infection, suggesting that these processes may compete for viral RNA.

Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5' untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation.

Several viral genome-linked proteins (VPgs) of plant viruses are intrinsically disordered and undergo folding transitions in the presence of partners. This property has been postulated to be one of the factors that enable the functional diversity of the protein. We created a homology model of Potato virus A VPg and positioned the known functions and structural properties of potyviral VPgs on the novel structural model. The model suggests an elongated structure with a hydrophobic core composed of antiparallel β-sheets surrounded by helices and a positively charged contact surface where most of the known activities are localized. The model most probably represents the fold induced immediately after binding of VPg to a negatively charged lipid surface or to SDS. When the charge of the positive surface was lowered by lysine mutations, the efficiencies of in vitro NTP binding, uridylylation reaction, and unspecific RNA binding were reduced and in vivo the infectivity was debilitated. The most likely uridylylation site, Tyr63, locates to the positively charged surface. Surprisingly, a Tyr63Ala mutation did not prevent replication completely but blocked spreading of the virus. Based on the localization of Tyr119 in the model, it was hypothesized to serve as an alternative uridylylation site. Evidence to support the role of Tyr119 in replication was obtained which gives a positive example of the prediction power of the model. Taken together, our experimental data support the features presented in the model and the idea that the functional diversity is attributable to structural flexibility.