The Identity of a Single Residue of the RNA-dependent RNA Polymerase of Grapevine Fanleaf Virus Modulates Vein Clearing Symptoms in Nicotiana benthamiana.

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 vein clearing symptoms 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 symptoms as GFLV-F13 RNA1 carrying K802 remained asymptomatic in N. benthamiana. No correlation was found between symptom expression and RNA1 accumulation, as shown by RT-qPCR. Additionally, the involvement of RNA silencing in vein clearing symptoms was ruled out by VIGS 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 (805 LLKT/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 vein clearing symptoms.