Graft-mediated PPV inoculation
To evaluate if RNA silencing-mediated resistance to PPV found in St5’-1,
St5’-6, and St5’-9 transgenic plums can be transmitted to apricots
grafted onto them, we utilized as inoculum source the Spanish PPV
isolate 3.3 RB/GF-IVIA (AF172346.1) belonging to the PPV-D strain
(García-Almodovar et al. , 2015), adopting two different virus
inoculation procedures.
First, wild-type (wt) apricot buds heavily infected by PPV were grafted
onto the transgenic plum rootstocks (Supplementary Figure 1), exposed to
a two-month artificial winter in the cold chamber, and then transferred
to the greenhouse. Eight weeks later, sprouted buds were evaluated for
virus resistance (see below). In the second experimental setting,
healthy apricot buds were grafted onto the transgenic plum rootstocks.
After an artificial winter and sprouting of the buds, the scions were
inoculated by chip-budding with new shoots of PPV-infected GF305 peaches
(Supplementary Figure 3). At the end of the cycle, plants
were again transferred to the cold chamber and evaluated for
virus resistance in the following cycle, as described above.