Vincenza Ilardi

Hairpin RNA-mediated silencing of Plum pox virus P1 and HC-Pro genes for efficient and predictable resistance to the virus.

We report the application of the hairpin-mediated RNA silencing technology for obtaining resistance to Plum pox virus (PPV) infection in Nicotiana benthamiana plants. Four sequences, covering the P1 and silencing suppressor HC-Pro genes of an Italian PPV M isolate, were introduced into N. benthamiana plants as two inverted repeats separated by an intron sequence under the transcriptional control of the Cauliflower Mosaic Virus 35S promoter. In a leaf disk infection assay, 38 out of 40 T0 transgenic plants were resistant to PPV infection. Eight lines, 2 for each construct, randomly selected among the 38 resistant plants were further analysed. Two hundred forty eight out of 253 T1 transgenic plants were resistant to local and systemic PPV infection. All transgenic single locus lines were completely resistant. These data indicate that the RNA silencing of PPV P1/HCPro sequences results in an efficient and predictable PPV resistance, which may be utilized in obtaining stone fruit plants resistant to the devastating Sharka disease.

Silencing of Plum pox virus 5′UTR/P1 sequence confers resistance to a wide range of PPV strains

An effective disease-control strategy should protect the host from the major economically important and geographically widespread variants of a pathogen. Plum pox virus (PPV) is the causal agent of sharka, the most devastating viral disease of Prunus species. We have shown previously that the hairpin RNA expression driven by h-UTR/P1, h-P1/HCPro, h-HCPro and h-HCPro/P3 constructs, derived from the PPV-M ISPaVe44 isolate, confers resistance to the homologous virus in Nicotiana benthamiana plants. Since the production of transgenic stone fruits and their evaluation for PPV resistance would take several years, the ISPaVe44-resistant plant lines were used to evaluate which construct would be the best candidate to be transferred to Prunus elite cultivars. To do that, nine PPV isolates of the D, M, Rec, EA and C strains originally collected from five Prunus species in different geographical areas, were typed by sequencing and used to challenge the transgenic N. benthamiana lines; 464 out of 464 virus-inoculated plants of lines h-UTR/P1, h-HCPro and h-HCPro/P3 showed complete and long-lasting resistance to the seven PPV isolates of D, M and Rec strains. Moreover, the h-UTR/P1 plants were also fully resistant to PPV-C and -EA isolates. Our data suggest that the h-UTR/P1 construct is of particular practical interest to obtain stone fruit plants resistant to the sharka disease.

Genetically engineered resistance to Plum pox virus infection in herbaceous and stone fruit hosts.

Plum pox virus (PPV), a Potyvirus, is the causal agent of sharka, the most detrimental viral disease affecting stone fruittrees. This review focuses on research carried out to obtain PPV- resistant transgenic plants and on how biotechnological strategies evolved in light of the scientific advances made during the last several years. Successful RNA silencing strategies that confer high level of resistance to strains of PPV have been developed and tested under laboratory and greenhouse conditions. Moreover, field tests showed that transgene-mediated RNA silencing was effective in protecting plum plants against aphid-mediated PPV infection. The new emerging biotechnological approaches for conferring PPV resistance are discussed.

Biotechnological strategies and tools for Plum pox virus resistance: trans-, intra-, cis-genesis, and beyond

Plum pox virus (PPV) is the etiological agent of sharka, the most devastating and economically important viral disease affecting Prunus species. It is widespread in most stone fruits producing countries even though eradication and quarantine programs are in place. The development of resistant cultivars and rootstocks remains the most ecologically and economically suitable approach to achieve long-term control of sharka disease. However, the few PPV resistance genetic resources found in Prunus germplasm along with some intrinsic biological features of stone fruit trees pose limits for efficient and fast breeding programs. This review focuses on an array of biotechnological strategies and tools, which have been used, or may be exploited to confer PPV resistance. A considerable number of scientific studies clearly indicate that robust and predictable resistance can be achieved by transforming plant species with constructs encoding intron-spliced hairpin RNAs homologous to conserved regions of the PPV genome. In addition, we discuss how recent advances in our understanding of PPV biology can be profitably exploited to develop viral interference strategies. In particular, genetic manipulation of host genes by which PPV accomplishes its infection cycle already permits the creation of intragenic resistant plants. Finally, we review the emerging genome editing technologies based on ZFN, TALEN and CRISPR/Cas9 engineered nucleases and how the knockout of host susceptibility genes will open up next generation of PPV resistant plants.

Identification, intra- and inter-laboratory validation of a diagnostic protocol for ‘Candidatus Liberibacter solanacearum’ in carrot seeds

Abstract‘Candidatus Liberibacter solanacearum’ (CaLsol) is a phloem-limited, unculturable, Gram-negative bacterium associated with emerging diseases in crops of the Solanaceae and Apiaceae families. As it has been shown to be seed-transmitted in carrot, emergency measures for exportation require carrot seed to be heat-treated or tested by PCR and found CaLsol free. Therefore, the identification and harmonization of a protocol for CaLsol diagnosis in carrot seed are becoming of socio-economic priority. We initially set up an improved DNA extraction method for Apiaceae seeds and identified, among the widely used PCR tests to detect and identify CaLsol, the real-time PCR developed by Li et al. (Journal of Microbiological Methods, 78(1), 59–65, 2009) and the end-point PCR by Ravindran et al. (Plant Disease, 95(12), 1542–1546, 2011) to be the most sensitive ones. The two PCR methods were initially intra-laboratory validated followed by a “Test Performance Study” involving 11 Italian laboratories that received both the samples and the material necessary to carry out the experiments. The results indicated that the improved DNA extraction method was robust and that the real-time PCR showed the highest analytical sensitivity in the intra-laboratory validation tests. Similarly, the real-time PCR outperformed the end-point PCR in the inter-laboratory comparison assay showing a higher percentage of accuracy, accordance, and concordance. The overall obtained data could be used for the appropriate application of phytosanitary measures against CaLsol.