Evaluation of transgenic Prunus domestica L., clone C5 resistance to Plum pox virus

Plum pox virus (PPV) is one of the most devastating diseases of Prunus species. Since few sources of resistance to PPV have been identified, transgene-based resistance offers a complementary approach to developing PPV-resistant stone fruit cultivars. C5, a transgenic clone of Prunus domestica L., containing the PPV coat protein (CP) gene, has been described as highly resistant to PPV in greenhouse tests, displaying characteristics typical of post-transcriptional gene silencing (PTGS). Moreover, C5 trees exposed to natural aphid vectors in the field remained uninfected after 4 years while susceptible transgenic and untransformed trees developed severe symptoms within the first year. In our study, a high and permanent infection pressure of PPV was provided by bud grafting of inoculum in the field trial of clone C5 conducted in the Czech Republic, in which PPV-infected and healthy control trees were used. Moreover, trees with combined inoculations by PPV, ACLSV and PDV were also used in the trial. The presence of the viruses throughout the tree tissues, the relative titre of the viruses and symptoms on C5 trees have been monitored over the years. The resistance stability of C5 clones under permanent infection pressure is discussed.Keywords: PPV, C5, resistance, real-time PC

Hairpin Plum pox virus coat protein (hpPPV-CP) structure in ‘HoneySweet’ C5 plum provides PPV resistance when genetically engineered into plum (Prunus domestica) seedlings

The genetically engineered plum ‘HoneySweet’ (aka C5) has proven to be highly resistant to Plum pox virus (PPV) for over 10 years in field trials. The original vector used for transformation to develop ‘HoneySweet’ carried a single sense sequence of the full length PPV coat protein (ppv-cp) gene, yet DNA blot analyses indicated that there was an inserted copy of the ppv-cp that appeared to be an inverted repeat structure. Since the resistance mechanism of ‘HoneySweet’ was found to be based on post-transcriptional gene silencing (PTGS), it was hypothesized that the inverted repeat structure conferred the resistance to PPV in ‘HoneySweet’. Sequencing of the transgene insertions confirmed the presence of an inverted repeat of the PPV-CP sequence. We hypothesized that transcription from this structure produced a hairpin (hp) RNA that was responsible for PTGS of the transgene and the destruction of PPV viral RNA resulting in the high level of resistance to PPV infection. In order to confirm this hypothesis the hpPPV-CP insert was cloned from ‘HoneySweet’ and transferred into ‘Bluebyrd’ plum seedlings through Agrobacterium tumefaciens transformation of hypocotyl slices. The introduced DNA contained the CP inverted repeat flanked by 35S promoters on either end. Transgenic plum plants containing single or multiple copies of this hp insert were inoculated with PPV D isolated from Pennsylvania, USA. PPV infection was evaluated through three cycles of cold-induced dormancy (CID) by symptom expression and by two or more ELISA and PCR tests. Of the 18 plants evaluated, eight were always virusfree, five occasionally had weak or moderate infections, and five plants were clearly infected in multiple tests. While all plants of some clones were virus-free others had a mix of uninfected and mildly infected plants of the same clone. Most of the resistant plants contained a single copy of the hp construct. These data strongly support the hypothesis that the hp structure of the ppv-cp insert in ‘HoneySweet’ plum can confer PPV resistance.Keywords: Breeding, gene silencing, Rosaceae, shark

Field trials of plum clones transformed with the Plum pox virus coat protein (PPV-CP) gene

Transgenic clones C2, C3, C4, C5, C6, and PT-6, of plum (Prunus domestica L.) transformed with the coat protein (CP) gene of Plum pox virus (PPV), PT-23 transformed with marker genes only, and nontransgenic B70146 were evaluated for sharka resistance under high infection pressure in field trials in Poland and Spain. These sites differed in climatic conditions and virus isolates. Transgenic clone C5 showed high resistance to PPV at both sites. None of the C5 trees became naturally infected by aphids during seven (Spain) or eight (Poland) years of the test, although up to 100% of other plum trees (transgenic clones and nontransgenic control plants) grown in the same conditions showed disease symptoms and tested positively for PPV. Although highly resistant, C5 trees could be infected artificially by chip budding or via susceptible rootstock. Infected C5 trees showed only a few mild symptoms on single, isolated shoots, even up to 8 years post inoculation. These results clearly indicate the long-term nature and high level of resistance to PPV obtained through genetically engineered resistance

RNAi to silence the Plum pox virus genome

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

RNAi to silence the Plum pox virus genome

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

Binding of ribosomes to the 5' leader sequence (N = 258) of RNA 3 from alfalfa mosaic virus.

RNA 3 of alfalfa mosaic virus (AlMV) contains information for two genes: near the 5' end an active gene coding for a 35 Kd protein and, near the 3' end, a silent gene coding for viral coat protein. We have determined a sequence of 318 nucleotides which contains the potential initiation codon for the 35 Kd protein at 258 nucleotides from the 5' end. This long leader sequence can form initiation complexes containing three 80 S ribosomes. A shorter species of RNA, corresponding to a molecule of RNA 3 lacking the cap and the first 154 nucleotides (RNA 3') has been isolated. The remaining leader sequence of 104 nucleotides in RNA 3' forms a single 80 S initiation complex with wheat germ ribosomes. The location of the regions of the leader sequence of RNA 3 involved in initiation complex formation with 80 S ribosomes is reported