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TARGETED ENGINEERING OF STONE FRUIT TREE GENOMES FOR RESISTANCE TO SHARKA

Periodic Reporting for period 1 - TESS (TARGETED ENGINEERING OF STONE FRUIT TREE GENOMES FOR RESISTANCE TO SHARKA)

Reporting period: 2018-05-01 to 2020-04-30

Plant viruses cause an estimated 50 billion € loss worldwide per year. Viral diseases represent one of the most limiting factors in European crop production having negative effects on the quantity and quality of foodstuffs. The recent identification of plant factors required for virus infection, together with the development of functional genomic tools in several economically important crops, offer novel opportunities to protect crop plants against viral diseases. The scope of TESS is to capitalize on the latest biotechnological tools in order to manipulate the plant/virus interactions towards non-host resistance and to match the research outputs with fruit industry and breeding applications. TESS benefits from multidisciplinary research teams involving genomics, tissue culture, molecular biology, virology and plant breeding. It focuses on Prunus species for stone fruit trees. Our goal in TESS is to engineer Prunus susceptibility genes either to create, by genome editing, a collection of de novo non-functional susceptibility genes (i.e. Knocked-Out KO genes) or to target specific amino-acids which are relevant for compatible plant/virus interactions, without knocking-out the host gene(s), to avoid any side effect for the plant. They will later be combined with over-expressed flowering time regulator cassettes, to promote early flowering, shorten the juvenile phase and allow rapid selection of mutants in those perennial species. The originality of TESS is to test the generic mode of interference of plant viruses with their host during the infection and use this information to implement complex and durable resistance in perennial fruit tree species.
In the first period of TESS, we performed activities and staff exchange essentially for the first two workpackages of TESS as follows:
In WP1, we applied the CRISPR/Cas9 system to target Prunus susceptibility genes either to create a collection of de novo non-functional susceptibility genes (i.e. KO genes) or to target specific amino-acids relevant for compatible plant/virus interactions, without knocking-out the host genes. For this purpose, several sets of editing vectors have been produced and peach and plum cultivars have been transformed. Transgenic lines are currently under screening.
In WP2, we targeted the stone fruit tree flowering pathways through the expression or editing of florigens (TFL1, DOG1 etc…), both in peach and plum; or through the development of viral vectors can be used to transiently express flowering regulators in stone fruit seedlings.
The expected results of TESS are to deliver:
1. Engineered forms of susceptibility host genes to PPV infection as breeding alternatives for viral disease resistance in stone fruit trees
2. Early flowering stone fruit genitors that display shorter juvenile phase thus allowing the implementation of rapid selection of mutants in those perennial species.
3. Fruit trees combining in the last phase of the project both constructs for non-functional susceptibility and early flowering genes to deliver PPV-resistant, transgene-free, plant material.
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