Final Report Summary - INTEREST (Intercontinental and temporal research studies on transgene engineered in plums)
In order to assess the efficiency of plant biotechnology to tackle virus infection, European Union has actually the opportunity to develop research in that field. As an alternate to classical breeding techniques, genetically modified (GM) technology can be applied to perennial crops. INTEREST was set to study the same transgenic plums harbouring virus transgene in two different continents: Europe and the USA. Among the significant reason that a co-operation between European and American was needed, HoneySweet clone, recognised as a GM plum co-produced by American (USDA) and European (INRA), is the model plant that researchers joined their efforts to consensually provide useful information about silencing in perennial crops.
Since hundred years ago, sharka disease was found to be caused by plum pox potyvirus (PPV). Classified as a quarantine pest, PPV has caused losses of more than 10.000 million Euros in Europe. While the pathway of PPV into North America is unknown, PPV was detected in Pennsylvania in 1999. PPV can be spread through natural infection by aphid vectors or by men through grafting with infected materials. The requirements of natural sources of resistance in classical breeding techniques took a relatively high priority in stone-fruit industry. Unfortunately, the paucity of resistance sources did not offer the availability of any exploitable clones. Thus, the development of transgene technology gave the opportunity to scientists to challenge sharka disease through INTEREST contract.
Taken HoneySweet as a model, INTEREST raises the question through a comparative study of the molecular mechanisms involved between plants grown in an infected area (high containment greenhouse, Bordeaux, France or field natural conditions, Prague, Czech Republic) and those grown in a virus-free area (Kearneysville, W-V, USA). Different parameters including the virus, different plant genes, the four seasons and the environment were taken into account. Since the mid of 1990s, artificial assays with woody perennial plums required the use of susceptible rootstocks where we grafted the studied cultivars. HoneySweet plum and controls (transgenic or conventional plants) were thus propagated. Based on such protocol, HoneySweet plum differently behaved from other clones in confined greenhouse because PPV could not systemically spread in the scions. Related to virus challenging assays in field natural conditions in Czech Republic, no PPV symptoms were found.
To better understand such reaction, INTEREST refers about links between silencing and PPV resistance through DNA methylation. The occurrence of the RNA-guided specification of DNA methylation (RdDM) to produce dsRNA is among the common feature involved. Another enzyme is also involved, the DNA methytransferases that catalyze cytosine methylation and maintain methylation after each round of DNA replication. Comparative studies with DNA extracted from HoneySweet grown in any environmental conditions revealed the same pattern. After evaluating American weather service data no obvious correlations between temperature and methylation were observed.
INTEREST through silencing studies in plum have forged the knowledge about the molecular interactions between virus transgene and PPV. The durable resistance is conferred by the siRNA resulting from the silencing process occurring in HoneySweet plum. Similarly to field testing conditions in Praha (Czech republic), a few HoneySweet plants grown in high containment greenhouse showed a few leaves, within mild spots of PPV symptoms, located around the grafting point at 3 weeks after bud breaking. These studies about silencing mechanisms in a perennial plant growing in field for more than 20 years gave an opportunity to European stone-fruit growers to include an added-value in their breeding programme. In extending research with virus co-existing that can be only inoculated by men (Prague, Czech Republic), there are no differences in the intensity of PPV leaf symptoms in HoneySweet plum infected by grafting with PPV-Rec, PPV-Rec+PDV, PPV-Rec+ACLSV, and PPV-Rec+PDV+ACLSV. Perception of such phenotypes is enigmatic because, only a few plants, and not all, showed these at the early stage of bud-breaking, then they recovered. In addition, the growth of the non-inoculated HoneySweet control trees was more vigorous in comparison with those inoculated with PPV and the combinations with PDV and ACLSV in Prague. These may have been due in the whole or part to the competition by the extensive growth of infected-bud (IB) shoots growing from the inoculated HoneySweet trees.
INTEREST pointed out that the comparative fruit evaluation between GM HoneySweet and conventional trees has shown that both have the same composition. However it is clear that both fruits differed through the siRNA content that specifically triggers PPV RNA in HoneySweet plum. Methylated virus transgene linked with siRNA accumulation in resistant plums were studied extensively in plants growing either in Europe (in the presence or absence of PPV) or in the USA (virus-free area). Disregard to virus infection, silencing in perennial plants plays a big role in the virus transgene regulation. Similarly too, any other endogenous genes like the Madbox genes are involved in development and regulation responsiveness. INTEREST is showing great promise, because, in addition to economic benefits gained from combatting PPV, the environment should benefit through reduced use of the insecticides that are normally required to kill aphid vectors of PPV.