FUNCTIONAL ANALYSIS OF A PUTATIVE DOWNY MILDEW DEFENCE GENE IN GRAPEVINE: STABLE TRANSFORMATION OF VITIS VINIFERA WITH VITIS RIPARIA ATL2 AND CHARACTERIZATION OF THE ATL GENE FAMILY IN GRAPEVINE.

Grapevine is one of the most important fruit crop in the world, with a high economic impact mainly due to the wine production. The European Vitis vinifera L. produces high quality grapes but is prone to several pathogens, which cause significant losses to viticulture worldwide. Even if chemical control is available for most of the diseases, agro-ecological concerns pushed the scientific community to search for alternative methods, in order to reduce both the environmental and economic issues associated to pest control. In particular several wild species of the genus Vitis from North America and Eastern Asia exhibit various levels of natural resistance towards distinct pathogens. Despite these natural resistance sources have been used to in the past to produce first resistant hybrids, none of them reached complete growers acceptance because of adverse organoleptic features. A significant step forward was achieved in the 2007, when the Vitis vinifera L. genome was completely sequenced. Since then the deeper knowledge of genetic determinants of both resistance and berry quality traits boosted the grapes scientific community in facing the Vitis susceptibility mainly by two different approaches: marker associated selection (MAS) breeding and genetic transformation. This PhD project was focused on the second approach, in particular we carried out the stable transformation of grapevine in order to enhance its resistance towards Plasmopara viticola, the causative agent of downy mildew. Transcriptomic responses to P. viticola in both the susceptible V. vinifera and the resistant V. riparia have been previously studied by our research group. Eight genes, specifically induced only in V. riparia upon the infection process, were identified as similar to ATLs (Arabidopsis Toxicos en Levadura), a gene family known to be rapidly induced by common elicitors. VrATL2, an ortholog of Arabidopsis thaliana ATL2, was selected as a promising candidate gene for stable grapevine transformation. As the ALT gene family was almost unknown in grapevine, the first step was the complete survey of the family members. The canonical RING-H2 domain was used as bite to search for putative ATLs within the translated genome of V. vinifera cv. Pinot Noir by PSI-Blast analysis. The resulting protein family was manually curated and analysed for specific molecular characteristics, phylogenesis and gene expression profiles in different grapevine tissues and developmental stages. Once set up the induction and maintenance of grapevine embryogenic material, we stably transformed V. vinifera cv. Shiraz producing plants with increased constitutive expression of VrATL2. The newly generated plants were molecularly characterised by Southern blot and Real Time qPCR analyses in terms of number of insertions and actual level of transgene expression respectively. The phenotyping of transformed plants for their resistance against P. viticola was carried out by means of two different methods: microscopical visual inspection and computational image analysis. The observed phenotype was further described analysing the transcriptomic changes in three selected transgenic lines by a microarray experiment and finally, on the same lines, the infection process was evaluated by microscopic observations of a time-course experiment. The last part of this PhD project was focused on the characterization of the ALT2 regulative regions from V. vinifera and V. riparia. After the bioinformatic analysis of the isolated regulative regions in terms of promoter structure and cis-acting elements, their ability to promote the transcription in heterologous systems was verified, by transient transformation of Nicotiana benthamiana and stable transformation of Arabidopsis thaliana. Lastly, we implemented the setup of a custom interrogable database of all cis-acting elements predicted for the ATL gene family in grapevine, which might facilitate further analysis within this family. In conclusion, this PhD project showed that even if stable transformation of grapevine remains an arduous and time-consuming task, and functional analysis are almost precluded, the accurate and informed choice of a candidate gene may provide good results in terms of expected phenotype. Moreover, the actual stable transgenic expression remains the most informative and plausible approach for functional analysis in V. vinifera plants. Indeed we were able to produce transgenic grapevines with enhanced resistance towards P. viticola by constitutive overexpression of the ATL2 gene. Despite further experiments are needed to confirm the durability of the observed resistance and to describe the associated phenotypic features over the time, we hope that in the future this approach could help in terms of sustainable agriculture and food safety.