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.