Conventional breeding, genome editing and microbial consortia to enhance sustainability and resilience of grapevine cultivation

In the present PhD thesis, we exploit different complementary approaches for a more sustainable control of grapevine diseases: inheritance of resistance genes, genome-editing to knock-out susceptibility genes and application of plants associated microorganisms to improve plants immunity. In the second chapter a review article describes in detail the most recent biotechnological approaches for crop protection, including genome editing, cisgenesis, RNAi and epigenetics. In the third chapter were reported the activities concern the conventional breeding approach. In this section the elite cv. Glera was crossing with resistant hybrids of different geographical origins. First the offsprings were evaluated for resistance loci presence. Then, eight plants carrying resistant genes to powdery and downy mildews were characterized by gas chromatography-mass spectrometry analyses to provide chemical nature of wine aroma. The fourth chapter described the activities concern the genome-editing application to knock-out susceptibility genes involved in powdery mildew interactions and in general to control the biotrophic pathogenic fungi. In details a novel CRISPR/Cas9 system based on specific recombinase (Cre/loxP) was developed with the aim of removing the ‘entire’ T-DNA cassette. In parallel, a review article about the application of Synthetic Community is presented in Chapter five. Finally, in Chapter six the exploitation of plant immune system is presented. The study aimed at clarifying the effects of arbuscular mycorrhiza priming on the grapevine growth-defence tradeoff

"Clean" genome editing in grapevine (Vitis ssp.)

In recent years new plant breeding techniques (NPBT), and in particular genome editing via Crispr/Cas9, emerged as breakthrough tools for the genetic improvments of agricultural species, allowing to precisely modify specific genes in shorter time compared to traditional breeding and without altering the genetic heritage of cultivars. Grapevine, the most economical valuable fruit crop in the world, may receive a major benefit from NPBT since viticulture is based on a few elite varieties. However, to date the European Coummission (EC) has not yet deliberated on the legal status of the NPBT products, whether they should or should not be covered by GMO legislation (Directive 2001/18). Waiting for the EC decision, we applied the Crispr/Cas9 system in grapevine for the inactivation of the VvMLO7 gene which plays a key role in susceptibility to powdery mildew. Our "clean" strategy aims at leaving in th eplant genome the minimal trace of exogenous DNA. It used the classical Agrobacterium tumefaciens (A.t.) to introgress Cas9, the sgRNA and the selection marker gene nptIIand allow removing the T-DNA cassette from the grapevine genome once the targeted mutations have been obtained. To this purpose, the Flp recombinase gene under the control of a heat-shock inducible promoter has been integrated in the T-DNA as well as its recognition sites (FRT), placed next to the A.t. left and right borders. NptII- and Cas9- positive lines of 'Chardonnay', 'Thompson seedless' and 'Microvine' were analyzed by next generation sequencing in order to assess the induced mutations in the target sites. Subsequently, the site-specific removal of the T-DNA cassette was evaluated in the heat-treated lines by quantifying nptII copy number with Real-time PCR method. The effect of powdery mildew infection on VvMLO7 edited plants in currently under evaluation

Modern biotechnological approaches toward sustainable viticulture

Conventional breeding does not allow the introgression of single traits without compromising the genetic background that characterize an elite cultivar. The exploitation of the new molecular techniques known as genome editing and cisgenesis make possible to modify or transfer single genes preserving all the characteristics selected with difficulty by breeders over a long-time span. To date, 27 QTLs have been associated with downy mildew disease resistance (Rpv1-Rpv27) and many of these have been employed in breeding programs for the introgression in genotypes of interest. Just in case of Rpv1 and Rpv3, however, the underlying genes have been identified and characterized. In both cases nucleotide-binding leucine rich repeat (NB-LRR) genes are present, codifying for receptors that act as cytoplasmic pathogen sensors, triggering a signal transduction pathway for cell-death mediated defense at the infection site. One of the biggest drawbacks of traditional genetic engineered plants is represented by the presence of transgenes (often selection markers), usually perceived as unsafe by consumers. The cisgenic approach aims at circumventing this problem avoiding the presence of exogenous DNA, introducing only the desired trait by using native genes from Vitis species, interfertile with V. vinifera. Adopting this strategy, the already characterized resistance genes RPV3 and/or RPV1 will be introduced in some elite Vitis vinifera varieties, highly appreciated by the wine industry. The outcomes will reduce the agrochemicals needs and the risks associated with their use, increasing the profitability of the vineyard and consumers\u2019 appreciation. Flower tissues of Glera, Sangiovese and Pinot Nero have been collected from field and fruit cuttings grown plants and from fruiting cuttings and used as explants for the induction of somatic embryos. PCR products of candidate genes, including native promoter and terminator, will be cloned in a suitable vector and transformed into competent E.coli. The gene sequences will be then isolated by PCR and cloned into a binary vector engineered with an inducible excision system. Transient expression assays will be used to evaluate the efficacy of the candidate genes into the different genetic backgrounds of the selected cultivars. The gene construct will be used for the transformation of grapevine embryogenic calli through A. tumefaciens infection. Infected calli will be transferred on selective media for the induction and germination of somatic embryos . Regenerated plantlets will hence be checked for the presence and expression of candidate genes. For the removal of exogenous sequences, chemical or thermal induction of the excision system will be used. Absence of Agrobacterium and backbone sequences will also be checked by PCR on transformants. Resistance and susceptibility to downy mildew will be tested on available material of interest by leaf disc bioassay or whole leaves inoculation of in-vitro and/or acclimatisedacclimatized plantlets

New plant breeding technologies towards a more sustainable viticulture

European grapevine cultivars are highly susceptible to many pathogens that are managed through large pesticide use. Nevertheless, the European policies promote pesticide use reduction and new environmentally friendly methods for a more sustainable agriculture. In this framework, grapevine genetic improvement could benefit from New Plant Breeding Technologies. In order to reduce fungal susceptibility, we will produce knock-out plants from embryogenic calli using CRISPR/Cas9 technology. Studies in barley reported the acquisition of powdery mildew resistance by knocking out susceptibility genes belonging to the MLO (Mildew Locus O) family. In this study, our approach takes advantage from CRISPR/Cas9 technology to perform a multiple knockout of MLO genes. Among the 17 VvMLOs reported in grapevine we designed constructs to target VvMLO6 and VvMLO7. Golden Gate assembly was used to produce three different constructs (containing two guideRNAs for each gene) to knocking-out the targets singularly or by producing a double mutant. Usually, the genetic engineering techniques, mediated by A. tumefaciens, involve the insertion of exogenous selectable marker genes. These markers are required for selection of transgenic plants, but they are undesirable to be retained in commercial transgenic plants due to possible toxicity or allergenicity to humans and potential environmental hazard. To overcome these limits, we opted for a \u201cclean\u201d editing strategy developing an inducible excision system. This approach is based on a recombinase technology involving the Cre-loxP system from the P1 bacteriophage under a heat-shock inducible promoter to be activated once the editing event(s) will be confirmed. Obtainment of embryogenic calli is one of the main bottlenecks for application of CRISPR/Cas9: for two seasons, we collected inflorescences from Chardonnay, Glera, Microvine, Pinot Noir, Sangiovese cultivars and two rootstocks, 110 Richter and SO4, cultured and maintained in vitro up to embryo development and then used to perform Agrobacterium tumefaciens GV3101 mediated transformation

NPBTs FOR SUSTAINABLE VITICULTURE MANAGEMENT TO BIOTIC AND ABIOTIC STRESS

New plant breeding techniques (NPBTs) aim to overcome traditional breeding limits for plant improvement to biotic and abiotic stresses satisfying the European Policies requirements that promote chemical input reduction and a more sustainable agriculture. We decided to apply genome editing (via CRISPR/Cas9) focusing on susceptibility genes to control powdery mildew: we chosen to knock-out two genes belonging to MLO (Mildew Locus O) family: VvMLO7 and VvMLO6. The same approach was used to cope with abiotic stresses, in specifc drought, performing a knock-out of four genes, two belonging to GST (Glutathione S-Transferase) and two to PME (Pectin Methyl Esterase) gene families. In parallel to genome editing, we also applied cisgenesis to move the resistance locus RPV3-1 (Resistance to Plasmopara viticola) into economically important cultivars. This locus is formed by two di\ufb00erent genes that were inserted individually and in combination to evaluate their e\ufb00ects. One of the drawbacks linked to classical Agrobacterium tumefaciens mediated transformation is the insertion of unrelated transgene (e.g., antibiotic resistance). These markers are required for transgenic plants selection, but undesirable to be retained in commercial plants due to possible toxicity or allergenicity to humans and animals, in addition to their potential hazards for the environment. To overcome these limits, we exploit an inducible excision system based on a Cre-lox recombinase technology controlled by a heat-shock inducible promoter that will be activated once the transformation event(s) will be confrmed. Embryogenic calli of Chardonnay, Glera, Microvine, Pinot Noir, Sangiovese, were used in stable transformation with A. tumefaciens carrying the genome editing construct with the MLO-guideRNAs and the cisgenic construct carrying the two RPV3-1 genes. Embryogenic calli of rootstocks 110 Richter and SO4 were transformed with genome editing construct carrying GST and PME guideRNAs in two independent transformations. Regenerated embryos from all the transformation events are now under evaluation

Chitosan nanocarriers-mediated delivery of double-stranded RNA "in planta"

Agriculture is currently facing numerous challenges: the rapid rise of the world population, the consequent growth in food demand, the global decrease in crop yield. Particularly regarding the last issue, climate change is worsening the environmental stresses that commonly affect crops, and the use of resources – such as fertilizers and pesticides – is highly inefficient and pollutant. In this context, research is looking for new approaches to improve crop productivity by more efficient and environmentally friendly practices. It has been shown that nanomaterials are suitable for the development of cutting-edge technologies with the aim of improving the delivery of bioactive substances on plants and to promote their resistance to biotic and abiotic stresses. Among organic polymers, chitosan, if used in the nanoscale form, shows both properties; it can induce biological responses concerning plant defense against diseases and pathogen attack, and it is particularly suitable as a carrier for several molecules. Another innovative method for the defense of crops is the exploitation of the spray-induced gene silencing (SIGS) based on the activation of the so-called RNA-interference (RNAi). It involves exogenous double stranded RNAs (dsRNAs) targeting an essential pathogen gene, which trigger the RNAi pathway leading to the translational repression by degradation of target homologous mRNAs. In our case, the research aimed to verify the feasibility of dsRNA distribution on plant surface by means of functionalized chitosan nanoparticles (CH-NPs), thus allowing the protection of the doping agent and its efficient delivery. Here we show the preliminary results regarding the characterization of CH-NPs, their loading with dsRNAs and their interaction with the leaf surface of Nicotiana benthamiana plants. The effects of the dose-dependent distribution were analyzed by confocal microscopy upon incorporation of a fluorescent probe

Actinomycetes come to rescue of viticulture sustainability

A great number of recent researches have shown that Actinomycetes can be considered as promising beneficial bacteria improving the growth and the capacity of the plants to face up both biotic and abiotic stresses. However, very few studies have been performed about the use of Actinomycetes in grapevine and all of them have focused their attention just on those isolated from the soil. For this reason, we decided to isolate bacteria from grapevine wood tissues characterizing a collection of 42 isolates, 27 of which belonging to the Actinobacteria phylum. These bacteria have been isolated from twenty years old grapevines without visual symptoms of wood diseases and subsequently used in dual culture assay against the main grape pathogens such as B. cinerea and the etiological agents of esca syndrome. Additionally, we are analysing antifungal effects of diffusible and volatile organic compounds (VOCs) produced by the isolates that previously showed a promising antagonistic activity against fungal pathogens. More studies are now ongoing to better understand their potential ability to promote plant growth features both in vitro and in vivo. Indeed, we are testing these bacteria on in vitro grapevine plants and on rooted cuttings. In the vitro assay we are evaluating their individual capacity to promote plant growth measuring the number of leaves and the roots length. Regarding to the rooted cuttings experiment, we inoculated a microbial consortium consisting of five Actinomycetes and three bacteria belonging to different phyla. The aim of the experiment is to test the effect of these beneficial bacteria both alone and in combination with some mycorrhizae fungi. We are now collecting several physiological parameters of the rooted cuttings to confirm the ability of these bacteria to increase the plant growth and wellness. More studies are needed but preliminary results display an interesting attitude of these actinomycetes as promising tool to improve viticulture sustainability.

Potential of New Plant Breeding Techniques for grapevine sustainability

New Plant Breeding Techniques (NPBTs) aim to overcome traditional breeding limits for plant improvement to biotic and abiotic stresses, satisfying the European Policies requirements that promote chemical input reduction and a more sustainable agriculture. Applying genome editing via CRISPR/Cas9 in grapevine, we focused our attention on susceptibility genes: to control powdery mildew we chosen to knock-out two genes belonging to MLO (Mildew Locus O) family VvMLO7 and VvMLO6. In parallel we also focused our attention on abiotic stresses, in particular drought stress, performing a knock-out of two different gene classes: I) two different isoforms of GST (Glutathione S-Transferase) that are involved in increasing of ABA (abscisic acid), lateral root branching and osmolytes accumulation; II) two different isoforms of PME (Pectin Methyl Esterase) involved in regulation of woody hydraulic proprieties modifying pit membrane thickness and porosity. In parallel to genome editing, we used cisgenesis to move the resistance locus RPV3-1 (Resistance to Plasmopara viticola) into economically important grape cultivars. This locus is formed by two different genes, TNL2A and TNL2B, that were inserted together (with native promoters and terminators). To avoid one of the drawbacks linked to classical Agrobacterium tumefaciens mediated transformation, hence the insertion of unrelated transgene, we exploited an inducible excision system based on a Cre-Lox recombinase technology. The system is controlled by a heat-shock inducible promoter that will be activated once the transformation event(s) will be confirmed allowing the removal of CRISPR/Cas and selection markers both in genome editing and cisgenic approach

HARNESSING GENOME EDITING TO ELUCIDATE THE ROLE OF HISTONE VARIANT H2A.Z IN GRAPEVINE

Plants continually encounter various environmental stresses that represent significant challenges to their growth, development, and survival. In particular, grapevine, which is a perennial plant of great economic importance, is subjected to both abiotic stresses, such as drought and extreme temperatures, and biotic stresses, including diseases like powdery (Erysiphe necator) or downy (Plasmopara viticola) mildew. Lastly, due to a long history of breeding programs, crops have developed the "domestication syndrome". This involves an imbalance in the plant use of resources towards growth and productivity, at the expense of resistance to pathogens and the ability to recruit beneficial microorganisms, such as mycorrhizae. Epigenetic modifications, such as DNA methylation and histone modifications, play an important role in stress responsiveness. Histone variants can be incorporated into nucleosomes in a targeted and replication independent manner thus influencing nucleosome structure and properties, gene transcription, DNA repair mechanisms and finally genome stability. Despite this, there is still much to be discovered about histone variants, especially on their role during plant-microorganism interactions. Currently, H2A.Z, one variant of the canonical histone H2A, seems to be the most involved in these mechanisms. Indeed, in plant, H2A.Z enrichment in the nucleosome occurs at Transcription starting site (TSS) of active genes and in the gene body of silent inducible genes. Plant mutants of histone H2A.Z variants that have been produced so far are viable, and therefore can reveal the function of these chromatin regulators throughout the life of the plant. Recent advances in genome editing techniques have provided powerful tools for precise modification of the genome. Our study aim is to investigate the application of genome editing (CRISPR-Cas9) techniques to modulate the expression of H2A.Z genes in grapevine and elucidate their role in in the interaction between plant and microorganisms, both beneficial and pathogenic. Target genes of interest for editing have been identified and transformation efforts are underway, aimed at both downregulation and overexpression of the H2A.Z genes. This will provide a wider knowledge of the role of H2A.Z in biotic interactions in grapevine