Symptom Severity of Nicotiana benthamiana Plants Inoculated with Agrobacterium Containing Infectious DNA-A Clones of Honeysuckle Yellow Vein Virus (HYVV)

To investigate the pathogenicity and virulence of the Honeysuckle yellow vein virus (HYVV) lacking betasatellites, PCR amplified unit-lengths of DNA-A genome of HYVV-[DJ] were cloned into binary vector pRI101-AN, and generated HYVV-[DJ]-1mer, -1.3mer and -2mer genomes. Each construct was transformed into Agrobacterium cells and agro-inoculated into young leaves of Nicotiana benthamiana. Except for the HYVV-[DJ]-1mer, HYVV-[DJ]-1.3mer and -2mer clones caused pronounced disease symptoms in N. benthamiana. HYVV-[DJ]-2mer agro-inoculated plants showed more severe plant stunting with downward leaf curling and crinkling than those of HYVV-[DJ]-1.3mer agro-inoculated plants. To discriminate the clone’s virulence quantitatively, SYBR Green-based real-time PCR was performed for the quantification of the target virulence gene DNA in agro-inoculated plants that were collected at weekly intervals for 4 weeks. Regression analysis was obtained from the standard curves by plotting Ct values over the logarithm of the amount of V1 protein gene DNA present in a dilution series of plasmid containing the full-length HYVV-[DJ] genome. Equation of the HYVV V1 DNA standard curve was used to quantify V1 gene DNA concentration in agro-inoculated plants with each clone. The accumulation of V1 gene DNA in HYVV-[DJ]-1.3mer agro-inoculated plants reached the peak level at 4 weeks post inoculation, while the accumulation of V1 gene DNA in HYVV-[DJ]-2mer agro-inoculated plants reached the peak level at 3 weeks post inoculation. The amount of V1 DNA in HYVV-[DJ]-1.3mer agro-inoculated plants was significantly more than that in HYVV-[DJ]-2mer agro-inoculated plants. Considering the results, there was a difference between the accumulation of virus DNA and the symptom severity of the analyzed plants agro-inoculated with each clone. It suggested that the infectious clones’ virulence is not necessarily correlated with the symptom severity.