Plum pox virus RNA silencing-mediated resistance can be transmitted from transgenic rootstocks to wild-type scions
To evaluate the ability of transgenic rootstocks to confer PPV resistance in wild-type (wt) apricot scions, as a first approach, we grafted buds from heavily PPV-infected wt apricots analyzing the PPV presence in the sprouting scions after an artificial winter (Supplementary Figure 1). Although many buds failed to sprout, probably due to the high virus load, PPV was not detected by RT-PCR in 75% and 40% of the scions sprouted from infected apricot buds grafted onto St5’-9 and St5’-1 rootstocks, respectively (Table 1) and in none of these rootstock lines. Conversely, PPV was detected in the new leaves of all susceptible St5’-7 rootstocks and all apricot scions. An intermediate PPV susceptibility behavior was observed for the tolerant St5’-6 line (Table 1). Collectively, more than 50% (five out of nine) of the wt scions sprouted from PPV-infected wt buds grafted onto the highly PPV-resistant transgenic rootstocks (St5’-9 and St5’-1) were PPV-free.
To corroborate the above results, a different inoculum procedure was envisaged to ensure the analysis of a more significant number of plants. To this end, the four transgenic plum rootstocks were grafted whenever possible with two-three healthy buds. After an artificial winter in the cold chamber, the buds were forced to sprout by severely trimming the rootstocks plants, and scions were PPV-challenged by chip-budding (Supplementary Figure 2). PPV infection was evaluated by RT-PCR after an artificial winter followed by sprouting in the greenhouse. When more than one bud sprouted on the same rootstock, all grafted apricots were evaluated independently. In those cases, the plant was considered resistant only when no one scion was PPV-positive by RT-PCR. One hundred and ten wt apricots grafted on transgenic rootstocks were analyzed (Figure 1). PPV was not detected in wt apricot scions of 48% of the St5’-9 rootstocks and 23% of the St5’-7 susceptible line (Figure 1). Twenty-eight plants were randomly chosen and re-evaluated at the beginning of the next cycle (about 2-3 weeks after the artificial winter) to confirm the data. We found that all results were identical except for one plant grafted onto a 5’-9 rootstock, one grafted onto a 5’-6, and two grafted onto a 5’-1 that switched from an RT-PCR positive reading to a negative one. The fact that in some RT-PCR positive scions, grafted onto PPV-resistant and -tolerant rootstocks, PPV was not detected more suggested a progressive recovery of wt scions from viral infection. To this end, all the PPV-positive plants in the first cycle were re-valuated after an additional 5-7 weeks (Figure 1, second cycle). Notably, in five plants from each line St5’-1, St5’-6, and St5’-9, that were PPV-positive at the first cycle, the virus was not detected again. Conversely, no one of the PPV-positive apricots plants grafted on the susceptible St5’-7 line became virus free. In addition, four of the five PPV-negative plants of the susceptible St5’-7 line became PPV-positive in the second cycle bringing the infection efficiency to 95,5% (Figure 1). The artificially inducing dormancy and greenhouse growth, followed by RT-PCR evaluation, was repeated for two additional cycles. In the fourth cycle, only 8%, 16%, and 28% of the wt apricots grafted on St5’-9, St5’-1, and St5’-6 were infected by PPV, respectively. The percent of PPV resistance observed on the wt scions correlates well with the transgenic rootstocks’ degree of PPV resistance (Table and Figure 1 and Garcia-Almodóvar et al., 2015). Our data support the notion that the RNA-silencing-based PPV resistance (García-Almodovar et al. , 2015) can be transmitted from PPV-resistant plum rootstocks to wt apricot scions and that its efficiency is augmented after successive growth cycles.