Genome editing is a plant breeding innovation that allows rapid targeted modification of plant genome, like natural mutations, to improve traits such as yield and disease tolerance. Loss-of-function mutations in one or more of ‘Mildew resistance Locus O’ (MLO) genes impart protection to plants from powdery mildew fungi and confer durable broad-spectrum resistance. This mlo-based resistance, detected initially as a natural mutation in barley, has been successfully employed for nearly four decades. Recently, mlo-based resistance was found to be engineered or identified, from natural mutants, in many plant species of economic relevance. Genome editing in grapevine is currently performed through conventional Agrobacterium[1]mediated plasmid delivery, which integrates foreign genes into the genome and is labeled GMO plants. A more recent CRISPR RiboNucleoProtein (RNP) delivery in protoplasts is a DNA-free approach but makes it difficult to regenerate plantlets and identify the gene-edited mutants. This project aims to establish an alternative gene-editing approach for the grapevine model to generate powdery mildew-resistant mutant vines free of foreign genes. We proposed to edit the MLO susceptibility genes through a combination of plasmid- and RNP-delivered CRISPR/Cas9 in microvine to produce DNA-free powdery mildew-resistant plants. The project is currently in the 3rd quarter of the final year, and the first phase of generating mlo mutants through conventional Agrobacterium-mediated gene editing has been concluded. MLOs 3, 4, 13, and 17 in grapevine have been identified as the closest orthologs of MLO genes responsible for powdery mildew resistance in other species. We obtained single mutants of candidate MLOs and the combinations of double and quadruple mlo mutants. The mutant combinations will help identify the appropriate MLO genes for mildew resistance in grapevine. Currently, the genome-edited mlo mutant embryos are at various stages of plantlet regeneration. Preliminary genotyping analyses of the transformants showed mutations in the predicted areas of the MLO genes targeted by the CRISPR system. So far, we have identified over 200 potential mlo mutants. The next phase of the project is the removal of the transgene cassette, integrated into the genome of mlo mutants by employing CRISPR RNP delivery to obtain transgene-free mlo vines. Towards this goal, we concluded the experimental work. We established 1) the methodology to purify the modified Cas9 protein, 2) the RNP complexations and in vitro cleavage tests, 3) the excision of the transgene cassette using in vitro RNPs, 4) the use Cell-Penetrating Peptides (CPP) to facilitate the entry of the CRISPR RNP into regenerable embryogenic cells, and 5) the optimization of the CPP-RNP delivery conditions. The editing of the target genes through the CPP-conjugated RNP delivery into the embryogenic cells was also tested on GFP-expressing embryogenic cell lines. We expect the regenerating mlo-edited plantlets to be ready for genotyping and powdery mildew resistance assays in 3 months, after which the second phase of the project (in vivo excision of the transgenic cassette) will begin to obtain transgene-free powdery mildew resistant vines.

Genome editing technology is a plant breeding innovation that allows rapid targeted modification of plant genome, similar to natural mutations, to improve crop traits such as yield and disease tolerance. Objective of this project is to apply CRISPR/Cas9 gene-editing to produce genome-edited non-transgenic (devoid of foreign genes) grapevine. We proposed to edit the susceptibility genes for powdery mildew (PM) in grapevine named ‘mildew locus O’ genes (MLO) through a combination of plasmid- and ribonucleoprotein (RNP)-delivered CRISPR/Cas9 into the intact embryonic cells to produce DNA-free PM-resistant grapevines. We adopted this two-phase gene-editing approach rather than RNP-delivery alone to overcome the large-scale screening logistics to identify mutated plants in the absence of selection marker genes.

* At the end of the 2nd quarter of Year 2, the conventional Agrobacterium-mediated transformation of microvine to generate mlo mutants has been concluded. Single, double and quadruple mutants of MLOs 3, 4, 13, and 17, involved in grapevine PM-susceptibility, are expected from the first phase gene-editing experiments. The CRISPR-Cas9 plasmid used for MLO gene editing was prepared with an alternative strategy which is explained in the following main text of the report. Gene edited mlo mutants are expected in 3-4 months after which characterization and identification of the specific MLO genes involved in grapevine PM-resistance and assessment of the level of resistance will be performed. As part of the second phase, removal of the transgene T-DNA cassette, integrated into the plant genome, by employing Cas9 RNP delivery has been proposed to generate transgene-free mlo mutant microvines. For that purpose, we plan to use Cell Penetrating Peptides (CPP) fused to Cas9 to facilitate the entry of the RNP into the embryogenic cells. Experiments to optimize the RNP delivery using CPP have continued during the 2nd year. After confirming the efficacy of the candidate CPP for cell penetration and internalization using fluorescent dye, we proceeded to test the delivery of CPP-conjugated Cas9 RNPsinto embryogenic cells. For these trials, GFP-expressing embryogenic callus has been used and the gene-editing rate of the GFP target gene has been assessed. Lower than expected editing rate was observed in these preliminary experiments and experiments to improve the editing rate and to optimize the methods to regenerate gene-edited embryos have been continuing in the year 2. When the MLO gene-edited microvines from first phase are ready, the RNP-delivery methods that are being optimized during the 2nd year will be employed to get the final transgene-free mlo mutant microvines.

The timing of ripening initiation is a major trait for wine grape production. Rapidly evolving climactic conditions will affect the ripening process of major cultivars in several growing regions of U.S, which may result in loss of fruit and wine quality. While the development of improved viticulture practices to mitigate effects of climate change is critical, the use of complementary approaches must be investigated. The identification of regulatory genes controlling the timing of ripening initiation is a research avenue to consider for molecular breeding programs in order to identify clones/cultivars more suitable to evolving climatic conditions. Innovative molecular practices in the field such as Spray Induced Gene Silencing have recently received scientific attention and its potential for rapid industrial application can be seen as alternate solution to traditional and molecular breeding. Yet, all these translational tools need scientific validation up front to characterize the cause-to-effect relationship between the gene and/or several genes and the trait of interest (fruit composition, disease resistance, etc.). The current research project aims to validate the contribution of a regulatory protein, VitviARF4, to the ripening initiation in grape berry. Three objectives were designed to achieve this goal; 1) the characterization of VitviARF4 via genetic engineering and the identification of its potential partners during the ripening process, 2) the identification of ripening-related genes that are targeted by VitviARF4, and 3) the evaluation of fruit composition on genetically engineered berries.

After the establishment of the microvine model at OSU to conduct the genetic engineering experiments, our research efforts were focused this year on several milestones of the project. For the objective 1, we conducted all the microvine transformations (four in total, control, two over-expression, and one knock – down) to either turn on or off the expression of VitviARF4. At least 20 independent transformed plants were selected per transformation event. We also confirmed protein-protein interactions of VitviARF4 with other proteins that play a major role in various physiological process of fruit ripening (sugar, brassinosteroids, ethylene, and epigenetic mechanism). For the objective 2, we demonstrated that we can control the expression of the transgene in transgenic microvines during the development of the plants. This result was critical to the success of the research outcomes of the objective 2. We have generated about 15 to 20 GFP positive transformed lines per construct and few of them were transferred to the greenhouse for being characterized. In parallel, we also conducted a Spray Induced Gene Silencing (SIGS) experiment on pre-véraison berries of V6 microvine. Our preliminary findings seem to support the delaying effect of VitviARF4 on the timing of berry ripening. We observed a faster ripening on berries treated with dsRNA aiming at silencing the expression of the endogenous VitviARF4. However, this experiment is currently repeated to confirm this first results. If confirmed, it might lead to a potential direct application of the SIGS technology in the field to manipulate trait. For the objective 3, we developed a
new analytical method to measure organic, amino, and phenolic acids, different types of carbohydrates, polyols, and three classes of flavonoids (anthocyanins, flavonols, and monomer and dimer of tannins). We have built an in-house library of 95 analytes that were tested against berry extracts from pericarp samples collected at different stages of grape berry ripening. We are currently testing the method on mature berries of the microvine and we have identified around 30 analytes covering the major families of compounds existing in grape berry. Dr. Tomasino has optimized the volatile and aroma analyses on mature fruits of regular microvines.