Ribosome profiles and riboproteomes of healthy and Potato virus A‐ and <i>Agrobacterium</i>‐infected <i>Nicotiana benthamiana</i> plants

Eskelin, Katri; Varjosalo, Markku; Ravantti, Janne J.; Mäkinen, Kristiina

doi:10.1111/mpp.12764

Cited by 17 publications

(19 citation statements)

References 69 publications

(142 reference statements)

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“…Polysomes were purified from PVA WD-Strep-RFP (OD 600 = 0.3) and PVA WT-Strep-RFP (OD 600 = 0.1) infected N. benthamiana plants (sampled at 4 dpi), via a Assymetric Field Flow Fractionation (AF4) protocol previously described in Eskelin et al (2019). (A) Upper panel presents AF4 elution profiles of representative PVA WD-Strep-RFP infected samples while the lower panel presents those of representative PVA WT-Strep-RFP infected samples.…”

Section: Author Summarymentioning

confidence: 99%

“…Many HCPro-induced transcriptional changes in infected plants occur via the RAV2 transcription factor [ 17 ]. Furthermore, HCPro interacts with ribosomes in PVA-infected plants [ 12 , 18 ]. In Ivanov et al [ 12 ], we suggested that polysome-associated high molecular weight complexes including HCPro and ARGONAUTE 1 (AGO1) may function in translational regulation [ 12 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

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Association of host protein VARICOSE with HCPro within a multiprotein complex is crucial for RNA silencing suppression, translation, encapsidation and systemic spread of potato virus A infection

Pollari

Varjosalo

et al. 2020

PLoS Pathog

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In this study, we investigated the significance of a conserved five-amino acid motif 'AELPR' in the C-terminal region of helper component-proteinase (HCPro) for potato virus A (PVA; genus Potyvirus) infection. This motif is a putative interaction site for WD40 domain-containing proteins, including VARICOSE (VCS). We abolished the interaction site in HCPro by replacing glutamic acid (E) and arginine (R) with alanines (A) to generate HCPro WD. These mutations partially eliminated HCPro-VCS co-localization in cells. We have earlier described potyvirus-induced RNA granules (PGs) in which HCPro and VCS co-localize and proposed that they have a role in RNA silencing suppression. We now demonstrate that the ability of HCPro WD to induce PGs, introduce VCS into PGs, and suppress RNA silencing was impaired. Accordingly, PVA carrying HCPro WD (PVA WD) infected Nicotiana benthamiana less efficiently than wild-type PVA (PVA WT) and HCPro WD complemented the lack of HCPro in PVA gene expression only partially. HCPro was purified from PVA-infected leaves as part of high molecular weight (HMW) ribonucleoprotein (RNP) complexes. These complexes were more stable when associated with wild-type HCPro than with HCPro WD. Moreover, VCS and two viral components of the HMW-complexes, viral protein genome-linked and cylindrical inclusion protein were specifically decreased in HCPro WD-containing HMW-complexes. A VPg-mediated boost in translation of replication-deficient PVA (PVA ΔGDD) was observed only if viral RNA expressed wild-type HCPro. The role of VCS-VPg-HCPro coordination in PVA translation was further supported by results from VCS silencing and overexpression experiments and by significantly elevated PVA-derived Renilla luciferase vs PVA RNA ratio upon VPg-VCS co-expression. Finally, we found that PVA WD was unable to form virus particles or to spread systemically in the infected plant. We highlight the role of HCPro-VCS containing multiprotein assemblies associated with PVA RNA in protecting it from degradation, ensuring efficient translation, formation of stable virions and establishment of systemic infection.

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Section: Author Summarymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Association of host protein VARICOSE with HCPro within a multiprotein complex is crucial for RNA silencing suppression, translation, encapsidation and systemic spread of potato virus A infection

Pollari

Varjosalo

et al. 2020

PLoS Pathog

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“…Overall, the regulatory system must ensure the safe packaging of the vRNA into virions when the amount of CP is sufficient. HCPro is an excellent candidate for the coordination of these functions as it participates in vRNA silencing suppression [2,3], VPg-mediated enhancement of vRNA expression [43], associates with ribosomes [44,45] and is required for particle formation (see Figure 3). The results of this study suggest that HCPro can deliver CP molecules to vRNA encapsidation, thus reducing translation inhibition (see Figure 5).…”

Section: Discussionmentioning

confidence: 99%

Interplay of HCPro and CP in the Regulation of Potato Virus A RNA Expression and Encapsidation

Saha

Lõhmus

Dutta

et al. 2022

Viruses

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Potyviral coat protein (CP) and helper component-proteinase (HCPro) play key roles in both the regulation of viral gene expression and the formation of viral particles. We investigated the interplay between CP and HCPro during these viral processes. While the endogenous HCPro and a heterologous viral suppressor of gene silencing both complemented HCPro-less potato virus A (PVA) expression, CP stabilization connected to particle formation could be complemented only by the cognate PVA HCPro. We found that HCPro relieves CP-mediated inhibition of PVA RNA expression likely by enabling HCPro-mediated sequestration of CPs to particles. We addressed the question about the role of replication in formation of PVA particles and gained evidence for encapsidation of non-replicating PVA RNA. The extreme instability of these particles substantiates the need for replication in the formation of stable particles. During replication, viral protein genome linked (VPg) becomes covalently attached to PVA RNA and can attract HCPro, cylindrical inclusion protein and host proteins. Based on the results of the current study and our previous findings we propose a model in which a large ribonucleoprotein complex formed around VPg at one end of PVA particles is essential for their integrity.

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“…Previously it has been proposed that r-protein isoform swapping between tissue types or during development might occur in plants (Giavalisco et al, 2005;Carroll et al, 2008). Evidence for different r-protein isoform compositions of ribosomes during phosphate-and iron-deficient stress in Arabidopsis (Wang et al, 2013), ultraviolet-B stress in Arabidopsis ((Falcone Ferreyra et al, 2013) and upon viral infection in Nicotiana benthamiana (Eskelin et al, 2019) have been proposed. Some level of alteration in ribosome composition during the stress treatments conducted here is evident in the significant changes in relative abundance of different r-protein isoforms following oxidative stress ( Figure 2B, Supplemental Table 2 for details).…”

Section: Discussionmentioning

confidence: 99%

Impact of oxidative stress on the function, abundance, and turnover of the Arabidopsis 80S cytosolic ribosome

Khidhir

Duncan

et al. 2020

The Plant Journal

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Abiotic stress in plants causes accumulation of reactive oxygen species (ROS) leading to the need for new protein synthesis to defend against ROS and to replace existing proteins that are damaged by oxidation. Functional plant ribosomes are critical for these activities, however we know little about the impact of oxidative stress on plant ribosome abundance, turnover and function. Using Arabidopsis cell culture as a model system we induced oxidative stress using 1µM of H2O2 or 5µM menadione to more than halve cell growth rate and limit total protein content. We show that ribosome content on a total cell protein basis decreased in oxidatively stressed cells. However, overall protein synthesis rates on a ribosome abundance basis showed the resident ribosomes retained their function in oxidatively-stressed cells. 15 N progressive labelling was used to calculate the rate of ribosome synthesis and degradation to track the fate of 62 r-proteins. The degradation rates and the synthesis rates of most r-proteins slowed following oxidative stress leading to an aging population of ribosomes in stressed cells. However there were exceptions to this trend; r-protein RPS14C doubled its degradation rate in both oxidative treatments. Overall we show ribosome abundance decreases and their age increases with oxidative stress in line with loss of cell growth rate and total cellular protein amount, but ribosome function of the aging ribosomes appeared to be maintained co-committently with differences in the turnover rate and abundance of specific ribosomal proteins. Data are available via ProteomeXchange with identifier PXD012840.

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Ribosome profiles and riboproteomes of healthy and Potato virus A‐ and Agrobacterium‐infected Nicotiana benthamiana plants

Cited by 17 publications

References 69 publications

Association of host protein VARICOSE with HCPro within a multiprotein complex is crucial for RNA silencing suppression, translation, encapsidation and systemic spread of potato virus A infection

Association of host protein VARICOSE with HCPro within a multiprotein complex is crucial for RNA silencing suppression, translation, encapsidation and systemic spread of potato virus A infection

Interplay of HCPro and CP in the Regulation of Potato Virus A RNA Expression and Encapsidation

Impact of oxidative stress on the function, abundance, and turnover of the Arabidopsis 80S cytosolic ribosome

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