Innovative RNAi Strategies and Tactics to Tackle Plum Pox Virus (PPV) Genome in Prunus domestica-Plum

Abstract

We developed an innovative RNAi concept based on two gene constructs built from the capsid gene (CP) cistron of the Plum pox virus (PPV) genome. First, designated as amiCPRNA, a potential molecule interfering with PPV genome translation and the second one is the ami-siCPRNA to target viral genome translation and PPV RNA replication. Following the previous engineering of these constructs in an experimental herbaceous host, they were introduced into Prunus domestica (plum tree) genome. Previously propagated onto a susceptible rootstock, these clones were graft-inoculated with PPV. After four dormancy cycles, and consistent with our experience of PPV infection, some clones showed a common phenomenon of silencing that can differ between the detailed plant phenotypes. Three different phenotypes were developed by the amisiCPRNA clones. First, the high resistance character shown by the amisiCPRNA plum-7 that was similar to the resistance expressed by HoneySweet plum. Secondly, a recovery reaction was developed by the two other amisiCPRNA plum-3 and plum-4 that differed from the rest, characterized as susceptible clones, among these were the amiCPRNA plums. Having assessed the behavior of these plums versus the herbaceous host accumulating the similar form of RNAi: ami-, si-, and ami-siRNA, challenging assays in perennials consistently reflect the natural context of viral genome targeting.

Robust Response to Plum pox virus Infection via Plant Biotechnology

“…Since an available number of replicates (3-6 copies) was obtained, plants were graft-inoculated prior to their transfer in cold for setting up an artificial dormancy. PPV-M was chosen to infect the clones because it causes more severe disease [4][5][6]16,17]. Initial testing for infection was based on experimental evidence for PPV infection through the appearance of symptoms (mosaic on BlueByrd plum and typical leaf distortion on peach) from 4 weeks after the first bud-break.…”

“…Plant genomic DNA of studied clones were extracted according to [16,17,21]. In total, 2 µg of DNA were digested overnight in parallel, with BfuCI and the isoschizomer MboI at 37 • C. After a precontrol of the digestion efficiency, one aliquote (1/10) of the digested DNA was amplified by PCR using the couple of primers 340 Fw and 660 Rev according to [16,17,22].…”

“…Plant genomic DNA of studied clones were extracted according to [16,17,21]. In total, 2 µg of DNA were digested overnight in parallel, with BfuCI and the isoschizomer MboI at 37 • C. After a precontrol of the digestion efficiency, one aliquote (1/10) of the digested DNA was amplified by PCR using the couple of primers 340 Fw and 660 Rev according to [16,17,22]. PCR conditions were one cycle of 94 • C, 2 min, 40 cycles of 94 • C, 30 s, 55 • C, 30 s, 72 • C, 1 min, followed by one cycle of 72 • C for 10 min prior to stop at 12 • C. In total, 1 Kb DNA weight marker (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), an aliquot of the amplified DNA was fractionated onto a 2.5% agarose gel electrophoresis.…”

“…Total RNAs were extracted according to [16,17,22,23]. 30 µg of the total RNA were loaded on 16% of a denaturing urea-PAGE.…”

“…To detect the PPV CP sequence either introduced or transcribed in plum genome, the use of a 32 P molecular probe is among the specific and reproducible system [16,17]. By PCR-amplifying the PPV CP sequence, we used, as template, the pGA482GG/PPVCP-33 recombinant plasmid [3] in a reagent mixture of 50 µL containing buffer, dATP, dGTP, dTTP and, α 32 P dCTP (Perkin Elmer, Waltham, MA, USA), 1U Taq DNA polymerase (Qiagen-Kit, Valencia, Hilden, Germany) with forward primer (CPFwd: AAGCTGAC-GAAAGACAGGACGAG) and reverse primer (RevCP: CTACACTCCCCTCACACCGAG-GAA).…”