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

AVF Proj. ID: 2320 / /Cat.: ,
Primary Investigator(s): Laurent Delucby

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

AVF Proj. ID: 1983 / /Cat.: ,
Primary Investigator(s): Laurent Delucby

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

AVF Proj. ID: 1657 / /Cat.: ,
Primary Investigator(s): Dario Cantuby

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.

EVALUATING TRAITS TO IMPROVE GRAPEVINE WATER-USE EFFICIENCY AND DROUGHT TOLERANCE

AVF Proj. ID: 2284 / /Cat.:
Primary Investigator(s): Megan K. Bartlettby

Summary: The goal of this multi-year project is to identify leaf and root traits that improve drought tolerance and water-use efficiency (WUE) in grape cultivars and rootstocks. Growers will need more drought tolerant and water-efficient vines to avoid declines in berry yield and quality under the hotter, drier conditions expected for California’s wine regions. This project is focused on leaf and root values for the drought tolerance traits the turgor loss point, measured in waterstressed conditions (TLPdry), and the adjustment in turgor loss point between well-watered and water-stressed conditions (DTLP, or TLPwet – TLPdry). TLP measures the water potential threshold that causes cell collapse, which impedes water transport through the leaves and roots. A more negative TLP indicates the leaves and roots can undergo more severe water stress before experiencing cell collapse. Plants can also change their cellular biochemistry to make TLP more negative under water stress, with a greater adjustment indicated as a larger DTLP. Previous studies found an important role for these traits in drought tolerance in other crop species, but these traits have never been assessed for their impact on grapevine performance under water stress. In the first year of this project, we evaluated whether rootstocks with a more negative root TLPdry and a larger DTLP allowed vines to maintain greater gas exchange (i.e., stomatal conductance, photosynthesis, and transpiration), canopy growth, and WUE under water stress. We also tested the ability of a rapid method previously developed for leaves to measure root TLP, to make it feasible to screen for this trait across the many genotypes developed in rootstock breeding efforts.

We subjected 8 commercially important rootstock varieties (420A, 5C, 101-14, Ramsey, Riparia Gloire, 1103, 110R, 140Ru), grafted onto the same scion (Chardonnay), to a greenhouse drought experiment, to evaluate the impacts of these traits on gas exchange, growth, and WUE. We found these rootstocks to vary significantly in TLP and DTLP. All of the rootstocks adjusted TLP to become significantly more negative under water stress, and rootstock differences in DTLP reversed their rankings in TLP between wet and dry conditions. The rapid method successfully captured the TLP variation measured by the more time-consuming standard method. Root TLPdry was significantly correlated with vine water use under water stress, in terms of stomatal conductance and whole-plant transpiration, but in the opposite direction than we expected. Instead, a less TLPdry was associated with greater water use, and there was no relationship with DTLP. Further, the rootstocks that growers identified as most drought tolerant in vineyard conditions also exhibited a less negative TLPdry. In contrast, photosynthesis was reduced by water stress, but was not related to any of the drought tolerance traits. Overall, these findings suggest that TLP plays an important role in rootstock drought tolerance, but differently than expected. Our future work will test whether cell collapse potentially benefits root growth by redistributing water and carbohydrates to support growth into wetter soil, and evaluate the impact of scion differences in leaf TLP and DTLP on vine drought tolerance.

Breeding, Genetics, and Germplasm Evaluation

AVF Proj. ID: 2183 / /Cat.:
Primary Investigator(s): Gan-Yuan Zhongby

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.

Evaluation of grapevine rootstock selections

AVF Proj. ID: 1668 / /Cat.:
Primary Investigator(s): Matthew Fidelibusby

The purpose of this project is to identify selections from a USDA rootstock breeding program that might warrant release as commercial stocks, and to develop useful data on the performance of recently released rootstocks from other breeding programs to aid growers in selecting appropriate stocks for their vineyards. For the past several years Dr. Gan-Yuan Zhong, USDA-ARS, screened seedling selections for resistance to aggressive strains of root knot nematodes and shipped cuttings of resistant selections to the UC Kearney Agricultural Center in Parlier, CA, where they were rooted and planted into a vineyard for observation. Thirty selections were sent in 2016, 40 in 2017, and the remaining 112 selections in 2018. The vines will be grown at Kearney for three years before being screened for their potential as rootstock mother vines. Cuttings of vines with excellent traits will be challenged with a multi-species inoculum of plant parasitic nematodes, in collaboration with Dr. Andreas Westphal. Genotypes showing excellent mothervine traits and superior resistance to root knot nematode and/or broad resistance to multiple parasitic nematode species, will be advanced to replicated field trials as grafted vines. Stocks that show excellent potential as grafted vines will be referred to the USDA for consideration of release.

Currently five selections (PC0333-5, PC0349-11, PC0349-30, PC04153-4, and PC0597-13), have advanced to a replicated trial in a commercial table grape vineyard in Delano, and six selections (the same five from Delano plus PC0495-51) are in a replicated trial in a commercial winegrape vineyard in Merced. The vines in Delano were planted in summer, 2016, and grafted to Autumn King in 2017, but many of the PC selections failed after budding and were thus replanted in 2018 and are becoming established. The same rootstock selections were planted in Merced in September 2016, but will not be grafted until 2019. The replanting in Delano, and the grafting in Merced, should resolve questions about the potential, if any, of these selections as young grafted vines. The Merced trial is adjacent to another rootstock trial planted by former UCCE advisor Lindsay Jordan in 2016. The second Merced trial includes full rows of 1103P, and more recently released stocks including RS3, RS9, GRN2, GRN3, GRN4, and GRN5 grafted to Malbec, and replicated four times. However, most vines on GRN5 failed, so GRN5 was eliminated from the trial. Vine training in this trial was mostly completed in 2018, and petiole and nematode samples were collected. These preliminary data suggest the various stocks may affect vine nutrition and that they might differ with respect to nematode resistance. It is anticipated that the first fruit quality and yield data will be collected from the Merced trial in 2019.

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

AVF Proj. ID: 1983 / /Cat.:
Primary Investigator(s): Laurent Delucby

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

AVF Proj. ID: 443 / /Cat.: ,
Primary Investigator(s): Andrew Walkerby

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.

An Integrated Approach to Understanding How Grapevine Root Systems Respond to and Recover from Drought Stress

AVF Proj. ID: 1847 / /Cat.:
Primary Investigator(s): Dr. Andrew J. McElroneby

With our first year of funding from CGRIC, we made significant progress on this project and exciting new discoveries about grapevine fine root responses to drought that shed light on the differences among genotypes/rootstocks. The start date of the project was delayed by ~12months while we waited for the funds to become available, thus we couldn’t justify submitting a renewal in January 2016 for additional funds. We are now submitting a renewal to continue these efforts, which will cover the costs for the second year of the project. Our labs are continuing to work together closely on this and other related projects. In addition to the experimental results described below, we have collected and begun analyzing suberin responses using flurol yellow staining for the following 16 rootstocks/genotypes from a drought experiment run by Dr. Kevin Fort (Walker Lab): 140RU, 101-14, 110R, Riparia Gloire, St. George, 1103P, Dog Ridge, 1616C, Ramsey, Freedom, Schwarzmann, Chardonnay, Cabernet Sauvignon, Colombard, GRN-1, and GRN-2.

We originally hypothesized that embolism formation would be would of the primary limiting factors reducing fine root conductivity (Lpr) under drought stress. Instead, we discovered that embolism formation is preceded by cortical lacuna formation (i.e. the fine root cortex is tearing apart under mild drought stress for tissue just behind the root tip), which coincides with a precipitous drop in Lpr. In a series of follow-up experiments using neutron radiography, we compared the growth dynamic responses of 110R (drought resistant) and 101-14 (drought susceptible) during drydown and recovery and also linked this to lacuna formation and Lpr. Surprisingly 110R exhibited more lacuna under mild drought stress that was linked to lower Lpr for this tissue despite less suberization and greater root elongation rates after drought stress. A preliminary similar patterns has been found in a comparison of Ramsey and Riparia Gloire. We now suspect that slower suberization rates for fine roots of drought resistant rootstocks may contribute to their greater susceptibility to drought induced cortical-lacuna, but this response could provide the adaptive advantage of ensuring that the root tip is receiving adequate water and other resources (i.e. internal carbon supply). In other words, the dysfunction created by the cortical lacuna (not in the tip, but in tissues starting ~2cm behind the tip would reduce the competing surface area of the root cylinder and ensure that any water that is available is being dedicated to continued growth at the tip. We will continue to pursue this and other related hypotheses with the proposed renewal submitted along with this report.

Determining the Role of Auxin-Response Factor 4 in the Timing of Ripening Initiation in Vitis Vinifera

AVF Proj. ID: 1983 / /Cat.:
Primary Investigator(s): Laurent Delucby

The timing of the ripening initiation is a major trait for wine grape production. Rapidly evolving climactic conditions might affect the ripening process of major cultivars in several growing regions of U.S resulting in loss of fruit and wine quality. While the development of improved viticulture practices to mitigate the effects of climate changes is critical, the use of complementary approaches should be investigated. The identification of genetic markers associated with the timing of ripening initiation will be valuable to develop targeted breeding programs aimed to tackle grapevine production in the context of climate changes. Innovative molecular practices in the field such as Spray Induced Gene Silencing have recently paid scientific attention and is seen as a potential industrial application. Yet, all these translational tools need a scientific study up front to validate the relationship between a gene and/or several genes with the trait of interest (fruit composition, disease resistance, etc.). The current research project aims to validate the role of a regulatory protein, VviARF4, on the timing of ripening initiation in grape berry. Three objectives were designed to achieve this goal; 1) the characterization of VviARF4 through genetic engineering and the identification of VviARF4 interactors during the ripening process, 2) the identification of ripening-related genes directly targeted by the activity of VviARF4, and 3) the evaluation of fruit composition on berries for which VviARF4 activity has been modified through the genetic engineering procedure.

Since June 2017 we focused our research efforts on several milestones of the current project. We established the microvine culture at OSU part of the objective 1. We conducted our first attempt for genetic engineering of the microvine. We successfully
generated embryogenic cells from immature floral buds, which is necessary to perform genetic transformation. For the same objective, we finalized our different genetic constructs to induce and silence VviARF4 in the microvine. Using a protein-protein interaction test, we identified 170 potential protein partners to VviARF4 and we are in the process of confirming some of these interactions by additional in vitro assays. Some of these interactors are very interesting because they are associated with ABA, ethylene
and sugar signaling pathway that are known to be important in the ripening process. For the objective 3, we adapted a new analytical method to measure metabolites associated with organic, amino, and phenolic acids, different types of carbohydrates, polyols, and three classes of flavonoids (anthocyanins, flavonols, and monomer and dimer of tannins). For those metabolites, we have built an in-house library of 95 analytes. We tested this library using berry extracts from pericarp samples collected at different stages of grape
berry ripening. We were able to identify around 30 analytes covering major families of compounds existing in grape berry. These include tartrate, malate, glucose, fructose, sucrose, and several polyphenol-related compounds. We will be using this analytical method for the aim 3 of this research proposal.