Identification of protein interactions of grapevine fanleaf virus RNA-dependent RNA polymerase during infection of Nicotiana benthamiana by affinity purification and tandem mass spectrometry

Osterbaan, Larissa J.; Hoyle, Victoria; Curtis, Michelle; DeBlasio, Stacy L.; Rivera, Keith; Heck, Michelle; Fuchs, Marc

doi:10.1099/jgv.0.001607

Cited by 4 publications

(16 citation statements)

References 32 publications

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“…Previous virus–plant studies provided a springboard for profiling the host transcriptome and proteome and biological pathway in GFLV–N. benthamiana interactions. − ,− Then, information from closely related plant viruses with similar phenotypes was considered to provide the following list of cellular pathways to probe: viral silencing, viral receptors, ,, kinase cascades, plant hormone synthesis, , integrated stress response, , ribosomal complexes, , and photosynthesis-related genes. , Searching relative terms to these seven pathways in the annotated N. benthamiana genome generated three pathways of interest for wildtype GFLV-GHu: (1) host defense, (2) ribosome filter hypothesis, and (3) post-transcriptional gene silencing (Figure ).…”

Section: Resultsmentioning

confidence: 99%

“…Raw files obtained from LC–MS/MS (73 samples/146 files total) were converted to .dat files with MSConvert from ProteoWizard . Peptide identification was then performed via Mascot Daemon (v2.5.1) against the previously defined “NibenNepo” database with N. benthamiana, GFLV, related nepoviruses, potential contaminants, and decoys to return protein abundance for analysis, as performed previously .…”

Section: Experimental Sectionmentioning

confidence: 99%

“…Host responses and cellular reprogramming that are induced during GFLV infection, particularly for symptom development, remain largely unknown. Current hypotheses attribute symptom eliciting responses to the GFLV RNA1-encoded RNA-dependent RNA polymerase (RdRP, protein 1E Pol) interacting with host components via protein–protein interactions . Previous attempts at identifying host proteins associated with GFLV 1E Pol were performed by affinity-purification MS using infectious clones carrying a V5 epitope-tagged 1E Pol .…”

Section: Introductionmentioning

confidence: 99%

“…Current hypotheses attribute symptom eliciting responses to the GFLV RNA1-encoded RNA-dependent RNA polymerase (RdRP, protein 1E Pol) interacting with host components via protein–protein interactions . Previous attempts at identifying host proteins associated with GFLV 1E Pol were performed by affinity-purification MS using infectious clones carrying a V5 epitope-tagged 1E Pol . Several host protein candidates were found to complex with GFLV 1E Pol ; however, a large gap remains for understanding how GFLV mechanistically elicits symptoms in infected plant hosts …”

Section: Introductionmentioning

confidence: 99%

“…Previous attempts at identifying host proteins associated with GFLV 1E Pol were performed by affinity-purification MS using infectious clones carrying a V5 epitope-tagged 1E Pol . Several host protein candidates were found to complex with GFLV 1E Pol ; however, a large gap remains for understanding how GFLV mechanistically elicits symptoms in infected plant hosts …”

Section: Introductionmentioning

confidence: 99%

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Profiling Plant Proteome and Transcriptome Changes during Grapevine Fanleaf Virus Infection

et al. 2023

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Viruses can elicit varying types and severities of symptoms during plant host infection. We investigated changes in the proteome and transcriptome of Nicotiana benthamiana plants infected by grapevine fanleaf virus (GFLV) with an emphasis on vein clearing symptom development. Comparative, time-course liquid chromatography tandem mass spectrometry and 3′ ribonucleic acid sequencing analyses of plants infected by two wildtype GFLV strains, one symptomatic and one asymptomatic, and their asymptomatic mutant strains carrying a single amino acid change in the RNA-dependent RNA polymerase (RdRP) were conducted to identify host biochemical pathways involved in viral symptom development. During peak vein clearing symptom display at 7 days post-inoculation (dpi), protein and gene ontologies related to immune response, gene regulation, and secondary metabolite production were overrepresented when contrasting wildtype GFLV strain GHu and mutant GHu-1EK802G Pol. Prior to the onset of symptom development at 4 dpi and when symptoms faded away at 12 dpi, protein and gene ontologies related to chitinase activity, hypersensitive response, and transcriptional regulation were identified. This systems biology approach highlighted how a single amino acid of a plant viral RdRP mediates changes to the host proteome (∼1%) and transcriptome (∼8.5%) related to transient vein clearing symptoms and the network of pathways involved in the virus–host arms race.

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Section: Resultsmentioning

confidence: 99%

Section: Experimental Sectionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Profiling Plant Proteome and Transcriptome Changes during Grapevine Fanleaf Virus Infection

et al. 2023

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Grapevine Fanleaf Virus RNA1-Encoded Proteins 1A and 1B^Hel Suppress RNA Silencing

Choi

Pakbaz

Yepes

et al. 2023

MPMI

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Grapevine fanleaf virus (GFLV, genus Nepovirus, family Secoviridae) causes fanleaf degeneration, one of the most damaging viral diseases of grapevines. Despite substantial advances at deciphering GFLV-host interactions, how this virus overcomes the host antiviral pathways of RNA silencing is poorly understood. In this study, we identified viral suppressors of RNA silencing (VSRs) encoded by GFLV using fluorescence assays and tested their capacity at modifying host gene expression in transgenic Nicotiana benthamiana expressing enhanced green fluorescent protein (EGFP). Results revealed that GFLV RNA1-encoded protein 1A, for which a function had yet to be assigned, and protein 1BHel, a putative helicase, reverse systemic RNA silencing either individually or as a fused form (1ABHel) predicted as an intermediary product of RNA1 polyprotein proteolytic processing. The GFLV VSRs differentially altered the expression of plant host genes involved in RNA silencing, as shown by RT-qPCR. In a coinfiltration assay with an EGFP hairpin construct, protein 1A upregulated NbDCL2/4 and NbRDR6, and proteins 1BHel and 1A+1BHel upregulated NbDCL2/4, NbAGO1/2 and NbRDR6, while protein 1ABHel upregulated NbAGO1 and NbRDR6. In a reversal of systemic silencing assay, protein 1A upregulated NbDCL2 and NbAGO2, and protein 1ABHel upregulated NbDCL2/4 and NbAGO1. This is the first report of VSRs encoded by a nepovirus RNA1 and of two VSRs that act either individually or as a predicted fused form to counteract the systemic antiviral host defense, suggesting that GFLV might devise a unique counter-defense strategy to interfere with various steps of the plant antiviral RNA silencing pathways during infection.

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A single viral amino acid shapes the root system architecture of a plant host upon virus infection

Roy,

Fuchs

2024

BMC Microbiol

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Background Grapevine fanleaf virus (GFLV) is one of the most detrimental viral pathogens of grapevines worldwide but no information is available on its effect on the root system architecture (RSA) of plant hosts. We used two wildtype GFLV strains and their single amino acid mutants to assess RSA traits in infected Nicotiana benthamiana and evaluate transcriptomic changes in host root gene expression in replicated time course 3’RNA-Seq experiments. Mutations targeted the multi-functional GFLV-encoded protein 1EPol*/Sd, a putative RNA-dependent RNA polymerase and determinant of foliar symptoms in N. benthamiana plants. Results Plant infection with wildtype GFLV strain GHu and mutant GFLV strain F13 1EPolG802K, both carrying a lysine in position 802 of protein 1EPol*/Sd, resulted in a significantly lower number of root tips (-30%), and a significantly increased average root diameter (+ 20%) at 17 days post inoculation (dpi) in comparison with roots of mock inoculated plants. In contrast, the RSA of plants infected with wildtype GFLV strain F13 and mutant GFLV strain GHu 1EPolK802G, both carrying a glycine in position 802 of protein 1EPol*/Sd, resembled that of mock inoculated plants. Modifications of RSA traits were not associated with GFLV titer. Root tissue transcriptome analysis at 17 dpi indicated dysregulation of pattern recognition receptors, plant hormones, RNA silencing, and genes related to the production of reactive oxygen species (ROS). For wildtype GFLV strain GHu, RSA modifications were correlated with an abundant accumulation of ROS in the pericycle of primary roots at 7 dpi and the duration of vein clearing symptom expression in apical leaves. Dysegulation of a hypersensitive response was an overarching gene ontology found through enrichment analyses of 3’RNA-Seq data. Conclusions Our findings revealed the causative role of lysine in position 802 of protein 1EPol*/Sd in a novel RSA phenotype during viral infection and documented GFLV-N. benthamiana interactions at the root level based on (i) antiviral response, (ii) receptor mediated production of ROS, and (iii) hormone regulation. A correlation between above and below ground symptoms was reported for the first time in plants infected with wildtype GFLV strain GHu. Further work is warranted to test whether the modified RSA of a plant host might impact GFLV acquisition and transmission by the ectoparasitic dagger nematode Xiphinema index.

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Identification of protein interactions of grapevine fanleaf virus RNA-dependent RNA polymerase during infection of Nicotiana benthamiana by affinity purification and tandem mass spectrometry

Cited by 4 publications

References 32 publications

Profiling Plant Proteome and Transcriptome Changes during Grapevine Fanleaf Virus Infection

Profiling Plant Proteome and Transcriptome Changes during Grapevine Fanleaf Virus Infection

Grapevine Fanleaf Virus RNA1-Encoded Proteins 1A and 1B^Hel Suppress RNA Silencing

A single viral amino acid shapes the root system architecture of a plant host upon virus infection

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Identification of protein interactions of grapevine fanleaf virus RNA-dependent RNA polymerase during infection of Nicotiana benthamiana by affinity purification and tandem mass spectrometry

Cited by 4 publications

References 32 publications

Profiling Plant Proteome and Transcriptome Changes during Grapevine Fanleaf Virus Infection

Profiling Plant Proteome and Transcriptome Changes during Grapevine Fanleaf Virus Infection

Grapevine Fanleaf Virus RNA1-Encoded Proteins 1A and 1BHel Suppress RNA Silencing

A single viral amino acid shapes the root system architecture of a plant host upon virus infection

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Product

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Grapevine Fanleaf Virus RNA1-Encoded Proteins 1A and 1B^Hel Suppress RNA Silencing