Abstract

Summary 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.

Highlights

  • The ribosomal 40S subunit is composed of 18S ribosomal RNA and 33 ribosomal proteins (r-proteins)

  • We investigated the effects of two plant pathogens, A. tumefaciens and Potato virus A (PVA, family Potyviridae), on N. benthamiana riboproteome and translational activity

  • Extracts were pelleted by ultracentrifugation at 170 000 g (P170K samples) from healthy, Agrobacterium- and PVA-infected N. benthamiana plants and further fractionated by asymmetrical flow field-flow fractionation (AF4) to obtain the ribosome profiles

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Summary

Introduction

The ribosomal 40S subunit is composed of 18S ribosomal RNA (rRNA) and 33 ribosomal proteins (r-proteins). This number includes ribosome-associated receptor for activating C kinase 1 (RACK1) (Pisarev et al, 2008; Sengupta et al, 2004). The translation of r-proteins occurs in the cytoplasm and, subsequently, they are transported into the nucleolus for subunit assembly. The two subunits combine during translation initiation to form a translation-competent 80S ribosome. Global down-regulation of translation initiation, which causes alterations in the abundance of 40S and 60S subunits, monosomes and polysomes, may occur in various stress conditions (Bailey-Serres et al, 2009)

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