We used a reverse genetics approach to identify residue 802 (lysine, K) of the GFLV strain GHu RNA1-encoded RNA-dependent RNA polymerase (RdRp) as a modulator of vein-clearing symptom development in Nicotiana 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. Structure predictions for the RdRp suggested that residue 802 is flanked by strongly predicted stable secondary structures, including a conserved motif of unknown function (805LLKT/AHLK/RT/ALR814).
°Larissa showed a single amino acid of GFLV modulates disease symptoms
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