The identify of a Single Residue of the RNA-dependent RNA Polymerase of Grapevine Fanleaf Virus modulates vein clearing symptoms in Nicotiana benthamiana

Authors: OSTERBAAN, LARISSA - Cornell University; CHOI, JESSICA - Cornell University; KENNEY, JAIMIE - Cornell University; FLASCO, MADDISON - Cornell University; VIGNE, EMMANUELLE - Inland Northwest Research Alliance, Inra; SCHMITT-KEICHINGER, CORINNE - Inland Northwest Research Alliance, Inra; REBELO, ANA-RITA - Boyce Thompson Institute; Heck, Michelle; FUCHS, MARK - Cornell University

Submitted to: Molecular Plant-Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/7/2019
Publication Date: 5/22/2019
Citation: Osterbaan, L., Choi, J., Kenney, J., Flasco, M., Vigne, E., Schmitt-Keichinger, C., Rebelo, A., Heck, M.L., Fuchs, M. 2019. The identify of a Single Residue of the RNA-dependent RNA Polymerase of Grapevine Fanleaf Virus modulates vein clearing symptoms in Nicotiana benthamiana. Molecular Plant-Microbe Interactions. 32:7. https://doi.org/10.1094/MPMI-12-18-0337-R.
DOI: https://doi.org/10.1094/MPMI-12-18-0337-R

Interpretive Summary: Grapevine fanleaf virus (GFLV) is spread by a soil-dwelling nematode and infects grapevines. The virus causes symptoms in the leaves and lowers fruit quality; thus, it is a commercially important pathogen of grapes. Some strains, or variants, of GFLV cause symptoms while other GFLV strains do not cause symptoms. Nothing is known about how GFLV causes symptoms in plants, so comparing virus strains at the molecular level that cause symptoms to strains which do not cause symptoms should reveal the virus components that are responsible for symptoms. In this study, the model host plant Nicotiana benthamiana was used to compare two strains of GFLV, F13 which does not cause symptoms, to GHu, which produces typical GFLV symptoms. Results showed that a single amino acid difference in the virus protein responsible for virus replication in infected plants was sufficient to explain why GHu caused symptoms and F13 did not. This work provides a critical tool for plant breeders to develop novel grape varieties that are resistant to GFLV infection in future research.

Technical Abstract: The mechanisms underlying host plant symptom development upon infection by viruses of the genus Nepovirus in the family Secoviridae, including grapevine fanleaf virus (GFLV), are poorly understood. In the systemic host Nicotiana benthamiana, GFLV strain GHu produces characteristic symptoms of vein clearing in apical leaves, unlike other GFLV strains such as F13, which cause an asymptomatic infection. In this study, we expanded on earlier findings and used reverse genetics to identify residue 802 (lysine, K) of the GFLV-GHu RNA1-encoded RNA-dependent RNA polymerase (1EPol) as a modulator of vein-clearing symptom development in N. benthamiana. Mutations to this site abolished (K to G, A, or Q) or attenuated (K to N or P) symptom expression. Noteworthy, residue 802 is necessary but not sufficient for vein clearing, as GFLV-F13 RNA1 carrying K802 remained asymptomatic in N. benthamiana. No correlation was found between symptom expression and RNA1 accumulation, as shown by reverse transcription-quantitative polymerase chain reaction. Additionally, the involvement of RNA silencing of vein clearing was ruled out by virus-induced gene silencing experiments and structure predictions for protein 1EPol suggested that residue 802 is flanked by strongly predicted stable secondary structures, including a conserved motif of unknown function (805LLKT/AHLK/RT/ALR814). Together, these results reveal the protein nature of the GFLV-GHu symptom determinant in N. benthamiana and provide a solid basis for probing and determining the virus-host proteome network for symptoms of vein clearing.