RNAi to silence the Plum pox virus genome
Résumé
Sharka is a major disease threatening stone fruit production. Originally reported from the Balkan region of Europe, it is spread throughout the European continent and in other temperate areas of the world: Middle-East, Asia, Japan, North and South America and in parts of the North of Africa. Biotechnology is among the alternative solutions adopted to control Plum pox virus (PPV) infection. Following the success of the 'HoneySweet' plum, small RNA interfering (siRNAs) technologies are the approach to disrupt PPV replication. siRNA technologies via genetic engineering are explored in Prunus domestica, a natural host of PPV, to target the PPV genome. The engineered siRNA in plums was explored by challenging plants with the 4 major strains of PPV that are frequently found in orchards: D (originating from France), EA (from Egypt), M (from Greece) and Rec (from Slovakia). The effects of siRNAs were found to be virus specific because the doublet of 21- and 25-nucleotides was activated and no PPV strains broke the resistance. These studies conducted through more than 3 dormancy cycles pointed out that the resistant phenotype is stable. The intron-hairpin CP RNA construct demonstrated that silencing is an efficient strategy to control PPV infection. These silencing studies were extended through the development of a novel approach to silence the PPV genome: the use of artificial miRNA. Combining the knowledge about the PPV sequence and the results of the computational target predictions, two gene constructs designed as amiCPRNA and amisiCPRNA were engineered in Nicotiana benthamiana model plants. The combined effects of ami-and si-RNAs were found successful to silence the PPV genome because more than 70% of tested clones were resistant. While little is known about the cooperative function of ami- and si-RNAs, our findings show that these RNAi technologies can be successfully applied to inhibit PPV replication in these model host-plants.