Transgenic-derived 22 and/or 24 nt PPV siRNAs accumulate in wild-type apricot scions grafted on the PPV-resistant transgenic plum lines.
The production of transgene-derived virus-specific siRNAs is a pre-requisite for RNA silencing to exert its virus-interference function. Moreover, the ability of the transgenic rootstocks to confer virus resistance to a grafted wt scion is dependent on the accumulation in the scions of the transgene-derived siRNAs above a certain threshold level (Sidorova et al. , 2021). To this end, we evaluated and compared the presence of the h-UTRP1-derived PPV siRNAs in the four transgenic plum rootstocks, the PPV highly resistant St5’-1 and St5’-9, the tolerant St5’-6, and the susceptible St5’-7 and in wt healthy apricot scions grafted on them.
Northern blot analysis identified two bands migrating slower than the 20 bp primer marker in the RNA extracted from all transgenic plum rootstock leaves. In particular, the highly resistant St5’-1 and St5’-9 plum lines and the tolerant St5’-6 line accumulated more PPV-specific 21-22 nt siRNAs compared to the PPV susceptible St5’-7 line (Figure 3). In addition, the St5’-6 rootstock accumulated 24 nt siRNAs. The detection limit of the northern blot did not permit to see the accumulation of siRNA, if any, in all the apricot scions. Leaves of apricot scions grafted onto most of the St5’-1 line (4 out of 7 of these plants), some of the St5’-9 and St5’-6 lines (2 out of 5 and 2 out of 6 plants, respectively) accumulated the 22 nt siRNAs alone or with 24 nt siRNAs, whereas 24 nt siRNAs were not detected in 7 plants analyzed from the apricot scions grafted onto the St5’-7 rootstock, but only the smaller 22 band was found in 3 of those plants (Figure 3). When seen, in the scions grafted onto the resistant St5’-1 and St5’-9 and the tolerant St5’-6 rootstocks, the signal for the 24 nt siRNAs was more intense than the 22 nt siRNAs indicating a preferential accumulation of the 24nt siRNAs in the recipient scions that was not found in the scions grafted on the susceptible St5’-7 rootstock.