Robust Response to Plum pox virus Infection via Plant Biotechnology.

Our goal was to target silencing of the Plum pox virus coat protein (PPV CP) gene independently expressed in plants. Clone C-2 is a transgenic plum expressing CP. We introduced and verified, in planta, the effects of the inverse repeat of CP sequence split by a hairpin (IRSH) that was characterized in the HoneySweet plum. The IRSH construct was driven by two CaMV35S promoter sequences flanking the CP sequence and had been introduced into C1738 plum. To determine if this structure was enough to induce silencing, cross-hybridization was made with the C1738 clone and the CP expressing but PPV-susceptible C2 clone. In total, 4 out of 63 clones were silenced. While introduction of the IRSH is reduced due to the heterozygous character in C1738 plum, the silencing induced by the IRSH PPV CP is robust. Extensive studies, in greenhouse containment, demonstrated that the genetic resource of C1738 clone can silence the CP production. In addition, these were verified through the virus transgene pyramiding in the BO70146 BlueByrd cv. plum that successfully produced resistant BlueByrd BO70146 × C1738 (HybC1738) hybrid plums.

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

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.

Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP(®) using reverse transcription-recombinase polymerase amplification.

Plum pox virus (PPV) causes the most destructive viral disease known as plum pox or Sharka disease in stone fruit trees. As an important regulated pathogen, detection of PPV is thus of critical importance to quarantine and eradication of the spreading disease. In this study, the innovative development of two AmplifyRP(®) tests is reported for a rapid isothermal detection of PPV using reverse transcription-recombinase polymerase amplification. In an AmplifyRP(®) test, all specific recombination and amplification reactions occur at a constant temperature without thermal cycling and the test results are either recorded in real-time with a portable fluorescence reader or displayed using a lateral flow strip contained inside an amplicon detection chamber. The major improvement of this assay is that the entire test from sample preparation to result can be completed in as little as 20min and can be performed easily both in laboratories and in the field. The results from this study demonstrated the ability of the AmplifyRP(®) technique to detect all nine PPV strains (An, C, CR, D, EA, M, Rec, T, or W). Among the economic benefits to pathogen surveys is the higher sensitivity of the AmplifyRP(®) to detect PPV when compared to the conventional ELISA and ImmunoStrip(®) assays. This is the first report describing the use of such an innovative technique to detect rapidly plant viruses affecting perennial crops.

Role of the 25-26 nt siRNA in the resistance of transgenic Prunus domestica graft inoculated with plum pox virus.

The reaction of a genetically engineered plum clone (C5) resistant to plum pox virus (PPV) by graft inoculation with the virus was evaluated. The resistance in this clone has been demonstrated to be mediated through post-transcriptional gene silencing (PTGS). A single C5 plant out of 30 plants inoculated with PPV M strain by double chip-budding showed mild diffuse mosaic 'Sharka' symptom at the bottom section of the scion. The upper leaves of this PPV-infected C5 plant remained symptomless and the virus was not detected in them by either DAS-ELISA or RT-PCR. An RNA silencing associated small interfering RNA duplex, siRNA (21-26 nt), was detected in non-inoculated C5 plants and in the portions of inoculated C5 plant in which PPV could not be detected. In the PPV-infected portion of the C5 plant and in C6 PPV susceptible plants only the approximately 21-22 nt siRNAs was detected. Cytosine-methylation was confirmed in C5 plants both uninfected and showing PPV symptoms. The 25-26 nt siRNA normally present in C5 was absent in PPV-infected C5 tissues confirming the critical role of this siRNA in the resistance of clone C5 to PPV infection. We also show that this PPV infection was limited and transient. It was only detected in one plant at one of four post-dormancy sampling dates and did not appear to affect the overall PPV resistance of the C5 clone.