Developing an Efficient DNA-Free, Non-Transgenic Genome Editing Methodology in Grapevine

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.

Breeding, Genetics, and Germplasm Evaluation

The overall objective of this research project is to develop and use a genome editing technology for trait improvement of elite grape cultivars. In our 2018-2019 work, we successfully demonstrated the feasibility of editing a grape color gene, VvMybA1, by delivering a VvMybA1editing construct, p201H-MybA1Double1(1176/1180), into V. vinifera ‘Chardonnay’ embryogenic callus through stable Agrobacterium transformation. Subsequently in this project year, we focused on editing the grape color gene using a non-transgenic approach. Our non-transgenic approach is based on the facts that genome editing components (i.e. Cas9 and gRNA) on the T-DNA of a construct in Agrobacterium can transiently be expressed in target cells before degraded in or stably integrated into the host cell genome and the transiently expressed gene editing components, when taking place at a right time with appropriate strength, can result in successful editing of a gene in the host cell. By using GUS as a reporter gene, we observed that the transient expression of GUS in ‘Chardonnay’ embryogenic callus reached the peak after 2-3 days of co-incubation with Agrobacterium carrying the GUS construct. Based on the GUS observations, we co-incubated ‘Chardonnay’ embryogenic callus with Agrobacterium carrying the same construct p201H-MybA1Double1(1176/1180) we used in the 2018-2019 stable transformation for editing the VvMyb1A color gene. The co-incubation experiments were conducted for 2 and 3 days, respectively. High throughput sequencing revealed that editing took place in both 2- and 3-day incubations. Various types of editing were found, including deletions, insertions, and substitutions. Among the deletion events, single bp deletion was most frequent, as expected. Deletions involving multiple bps (2-15 bps) were also observed. Many of these events were likely resulted from transient expression of the CRISPR-Cas9 and VvMyb1A gRNAs. While the overall editing frequencies were very low (<0.015%), we demonstrated the success of editing a grape gene through transient Agrobacterium transformation. Now the key issue for grape gene editing is not so much about whether we can edit a grape gene in a non-transgenic manner, but about how to select the edited events from millions of non-edited cells. Solving this issue will be the focus of the proposed 2020-2021 project.

As a part of this continuing project, we induced some transgenic vines from the embryogenic callus transformed with the p201H-MybA1Double1(1176/1180) construct in the previous project year 2018-2019. Unfortunately, the induced vines showed extremely low vigor in tissue boxes, likely due to some undesirable clonal variation in the batch of ‘Chardonnay’ callus used. New ‘Chardonnay’ callus transformed with the same construct was produced in this project year and transgenic vines from the callus will be induced. The purpose of generating stable transgenic vines from the experiment is to demonstrate the phenotypic effect after the transposon Gret1 is removed from the VvMybA1 gene in ‘Chardonnay’.

Developing an efficient DNA-free, non-transgenic Genome editing methodology in grapevine

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 bacterial selection markers.

Since the start of project in July 2019, the CRISPR plasmid to be used in the first phase of MLO gene editing is being built. To deliver CRISPR-Cas9 RNP in the second phase, we planned to use cell penetrating peptides (CPP) fused to Cas9 to facilitate Cas9 entry into the embryogenic cells. Experiments to optimize the methods for RNP delivery using CPP and to identify the optimum stage of embryogenic callus, efficiency of the CPP to penetrate the cells, and the treatment methods have been undertaken. The CPP has been synthesized and the efficiency of cell penetration and internalization were assessed using fluorescent dye attached to the CPP. Based on the detection of the dye in majority of the cells in the treated callus we confirmed that Cas9 protein can be delivered into embryogenic cells using this CPP. Methods for the bacterial expression and purification Cas9 protein and its conjugation with CPP have been optimized. Cleavage activity of the CPP-conjugated Cas9 protein is assessed through in vitro cleavage experiments using the guide RNA targeting GFP gene. In the optimization process, GFP-expressing embryogenic callus has been used to treat with the RNP in order to assess the efficiency and accuracy of RNP-mediated gene-editing. Data from these experiments will be used to streamline the process of MLO gene editing in microvine.

Determining the role of Auxin-Response Factor 4 in the timing of ripening initiation in Vitis vinifera

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.

Integrating systems biology with marker assisted selection to guide the stacking of powdery mildew resistance genes

The long-term goal of this project is to develop grape varieties that possess effective and durable resistance to powdery mildew (PM). Stacking resistance genes from multiple resistant genetic backgrounds and with the least functional redundancy is a proven breeding strategy to improve both durability and level of resistance. This strategy requires (a) the identification of multiple sources of resistance, (b) the functional characterization of the mechanisms of resistance to prioritize optimal genetic combination, and, finally, (c) marker assisted breeding to introduce the selected genes into elite varieties.

In continuation of our multi-year breeding effort, with the awarded budget in the 2017-2018 funding period we have continued the functional characterization of resistance responses activated in presence of known PM resistance loci. Analysis of the data generated by the experiments for the functional characterization of Ren2, Ren3, Ren4, Ren6, Ren7, Run1, Run1.1, Run2.1 and Run2.2 showed differences of gene expression between accessions in response to PM. Deep-learning analysis to predict the best R gene combination for gene stacking by marker assisted breeding is
ongoing.

Breeding, Genetics, and Germplasm Evaluation

The overall objective of this research project is to develop and apply a genome editing tool for trait improvement in grape cultivars. During 2018-2019, we built a CRISPR-Cas9 construct for modifying the color gene VvMybA1 and transformed it into V. vinifera ‘Chardonnay’ embryogenic callus via Agrobacterium transformation. We successfully demonstrated the efficacy of the editing machinery components in the construct and obtained transgenic callus containing cells with edited color gene VvMybA1. A 10-kb-long Gret1 transposon fragment in the promoter region of VvMybA1 in ‘Chardonnay’ was removed in the edited cells. Further analysis revealed 20-60 bp deletions at or near the target sites. The overall frequencies of these deletions/insertions were generally low, with the maximum being about 0.5%. From this work, we were the first to demonstrate the feasibility of removing a large piece of DNA from a locus in grapevine. We also demonstrated the technical feasibility for modifying the color gene, an important fruit quality trait. In parallel, we tried two new delivery methods for editing VvMybA1 and VvPDS genes in callus: a) bombardment of plasmid DNA for transiently expressing both Cas9 and gRNA components in grape embryogenic callus; and b) bombardment of in vitro preassembled Cas9–gRNA ribonucleoproteins into grape embryogenic callus. Our results showed that the direct delivery methods didn’t worked well, and the successful editing rate was too low to have any practical utility. This is not a surprise as no successful edited vines have been produced by direct delivery methods due to tremendous technical challenges. We will propose new approaches for improving the direct delivery methods in the project year 2019-2020.

Determining the role of Auxin-Response Factor 4 in the timing of ripening initiation in Vitis vinifera

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 induce the conditional expression of the transgene in transgenic
microvine plants. This result was critical to the success of the research outcomes of the
objective 2. 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
is currently optimizing the volatile and aroma analysis on mature fruits of the microvines.

Development of Next Generation Rootstocks for California Vineyards

We are making strong progress in streamlining the assay’s for nematode, salt and phylloxera screening to test new germplasm, existing breeding populations to single out best rootstock selections, and test breeding and mapping populations. We have dynamic duo of Becky Wheeler and post-doc Daniel Pap to greatly accelerate nematode screening efforts. We have built up inoculum to carry out germplasm screening for the dagger nematode. Salt screening of germplasm that was promising in earlier screens was initiated at higher concentrations (75mM) to select optimum accessions that we could use in crosses. At the same time, we are initiating salt screening of breeding populations with different accessions of V. longii and 140Ru to look for segregation in order to carry out marker development process. We are making good progress in better understanding of root architecture in multiple rootstock species including Vitis berlandieri. The key point from different drought screens is that specific root length and root diameter is key features that could be used to test rootstock selections. Trials of selected accessions that pass the screening for horticultural features, nematode, phylloxera and salt tolerance are in pipeline for 2018-2019 funding cycle.

Deep Sequencing for Trunk Disease Diagnostics

The aim of this multi-year project is to develop rapid and cost-effective diagnostic methods based on Next Generation Sequencing (NGS) technologies for detection, identification and quantification of trunk pathogens in asymptomatic and symptomatic grape wood. In the 1st year of the project (2015 – 2016) we collected diseased wood material from commercial vineyards and characterized the associated fungal pathogen species using traditional methods, such as morphological and sequence-based identification of purified fungal colonies (see progress report for the 2015-2016 funding cycle). We used these samples to determine how effective ITS-sequencing, meta-genome sequencing and metatranscriptome  sequencing approaches are in identifying and quantifying pathogenic species directly in planta. Data simulations allowed us to determine what mapping algorithm was the most specific and sensitive in detecting trunk pathogens both qualitatively and
quantitatively. All NGS methods we tested were in agreement with traditional diagnostic methods, but also allowed us to detect simultaneously multiple pathogen species with no
need of hands-on sample culturing and colony purification. Additionally, unlike traditional diagnostics, which are strictly qualitative, NGS approaches allowed us to determine the
relative abundances of the different infecting species. Among all methods tested, ITS-seq is still the most cost-effective until library preparation costs for RNA and DNA-seq do not decline significantly. For this reason, ITS-seq was chosen for further protocol optimization.
Both sensitivity and specificity of the ITS-seq approach remain to be improved for diagnostics purposes. In the second year of the project (2016-2017), we (a) confirmed that NGS allows the detection with high specificity of actively infecting pathogens when vines
are experimentally infected with individual pathogen strains; (b) established that NGS detection is quantitative and allows to differentiate between diseased and healthy vines; (c)
developed a protocol for testing dormant cuttings and started testing cuttings provided by a commercial nursery. In the 2016-2017 funding cycle, we also developed a new DNA extraction protocol that reduced the time required for processing and the amounts of
sample, reagents and waste. In the current funding cycle (2017-2018), we have (a) completed the analysis of trunk pathogen presence in propagation material obtained from nurseries; (b) began a collaboration with Dr. Urbez-Torres at Agriculture and Agri-Food Canada to expand the sampling of propagation material to two additional nurseries and compare directly our approach with their DNA-macroarray method and (c) developed a new set of optimized primers for ITS-seq designed specifically to target the ITS of grapevine trunk pathogens.

Targeted Genome Editing for Trait Improvement in Vitis vinifera

Targeted genome editing to modify gene expression has rapidly emerged as an effective tool for cultivar improvement. This approach can result in two types of new grape varieties with one or several trait improvements: 1) Transgenic clones of existing varieties, or 2) Non-transgenic breeding lines to develop new varieties. In this first phase of the project, we aimed to demonstrate this technology by enhancing the powdery mildew resistance of ‘Thompson Seedless.’ Among the approaches that confer powdery mildew resistance in Vitis vinifera, silencing of susceptibility genes has proven to be promising both in terms of efficacy as well as potential durability. We previously showed that reducing expression of grapevine VvPmr6A, VvPmr6B, VvMloA, or VvMloQuadX resulted in up to a 60%reduction of powdery mildew incidence, even though our artificial microRNA approach only partially reduced target gene expression. In this current work we are using a Transcription Activator-like Effector Nuclease (TALEN) strategy to completely knock out our target genes and obtain even stronger disease resistance. In addition, we have identified two additional candidates that are grapevine genes directly related to Arabidopsis Pmr4 and Pmr5, which confer strong powdery mildew resistance in other plants. In this context, in the past year we were successful in assembling 7 TALEN constructs targeting all 6 target genes. The constructs are now being transferred into the final plant destination vector, which is expected to be sent for grape transformation by late February 2015.

Based on our previous results and publications in other plants, we expect to see a significant increase in powdery mildew resistance. This targeted genome editing approach will demonstrate the potential to knock out genes in V. vinifera, establishing a protocol that could be extended to various traits in improving existing or new varieties. In the current and subsequent years, we aim to demonstrate another genome modification technique (dTALE-mediated activation of native gene expression) for increased abiotic stress tolerance in grapevine.