Sharka disease, caused by the Plum pox virus (PPV), is one of the most harmful, quarantine viral diseases that affect stone fruit crops. The absence of natural resistance to the virus in stone fruits has become a decisive factor for the use of genetic transformation methods to obtain stable forms. The eIF(iso)4G and eIF(iso)4E genes encode translation initiation factors used in the PPV life cycle. In the presented study, the effect of silencing these genes using the RNA interference method on the resistance of sour cherry rootstock 146-2 plants (Prunus pumila L. x Prunus tomentosa Thunb) to the sharka disease was studied. Two vectors have been created for the genetic transformation of plants, with self-complementary sequences of the eIF(iso)4G and eIF(iso)4E gene fragments. The hairpin expression cassette contains a strong promoter of the peach ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) gene, as well as an intron and terminator of the same gene. We used the pMF1 vector containing recombinase R and a codA-nptII gene which makes it possible to obtain intragenic marker-free plants. A successful genetic transformation was carried out by the AGL0 strain of A. tumefaciens. Whole leaves of shoots cultivated in vitro were used as a source of explants. Eight independent transgenic lines of rootstock 146-2 were obtained in experiments (sixlines with a hairpin to the eIF(iso)4G gene and two lines with a hairpin to the eIF(iso)4E gene). Their status was confirmed by the PCR and Southern blotting. The obtained plants were acclimatized in a greenhouse. The silencing of the eIF(iso)4G and eIF(iso)4E genes in transgenic plants was confirmed by the quantitative PCR. The presence of specific small interfering (si) RNAs was confirmed by the method of Northern blotting. Plants of all transgenic rootstock lines were infected with PPV by the method of grafting with infected buds. Resistance to the PPV infection of the obtained transgenic plants was carried out by using an enzyme immunoassay. The ELISA results showed that silencing the eIF(iso)4G gene did not lead to increased resistance while silencing the eIF(iso)4E factor gene led to increased resistance to the PPV, and the one line's plants showed no signs of infection for two years after infecting. The work demonstrates a (promising) approach in which the creation of stone cultures resistant to the plum pox virus can be achieved by suppressing the genes of translation initiation factors in clonal rootstocks.
Keywords: Prunus; RNA interference; clonal rootstock; gene silencing; plum pox virus; sharka; translation initiation factor.