Effector-assisted breeding against powdery mildew in grapes.

In last three decades, a great deal of effort and resources have been placed by the grape breeding community on the identification of resistance (R) loci against the grape powdery mildew (GPM) pathogen Erysiphe necator, and their introgression in grapevine varieties of interest. However, the success of these breeding programs can be put at risk, if R-loci that the pathogen can overcome are deployed in the field. Moreover, breeders currently lack the tools that would enable them to swiftly identify new sources of resistance, and predict their durability under field conditions. Effector- assisted breeding has proven a potent contributor to modern breeding for the identification, functional characterization, and deployment of R-genes. The basis of the effector-assisted breeding lays on the principle that dominant R-genes typically mediate resistance through the recognition of matching effector proteins from the pathogen. During the first year of the project, we took the first steps towards the development of an effector-assisted breeding program in grape. Specifically, efforts were made to develop an Agrobacterium tumefaciens-based assay for the transient expression of GPM effectors in select grapevine lines that are currently used at the UC Davis GPM resistance breeding program, thus enabling the functional profiling of matching effector and R- gene pairs. Using as bioreporter the gene encoding for the enhanced green fluorescent protein (eGFP), different combinations of binary plasmid vectors and A. tumefaciens strains were tested for their efficacy to transiently express eGFP in Nicotiana benthamiana, as this is a more amendable and easier to work with system. The best binary vector/A. tumefaciens strains combinations were then tested for their ability to transiently express eGFP in select grapevine lines. A protocol for vacuum infiltrating detached grapevine leaves instead of entire plantlets was developed in parallel, thereby increasing the assay’s practicality and capacity. To this end, we were able to work out the conditions for achieving good levels of agroinfiltration into detached grapevine leaves but the expression level of the transgenes in agroinfiltrated tissue remains low and currently undergoes further optimization. Nonetheless, a set of 35 effector-encoding genes were cloned in one of our select binary vectors that we further modified it to add a 6x-His tag at the C-terminus of each transiently effector and their transient expression was tested in N. benthamaina. For 30 of these effectors we were able to confirm expression in this plant species either by means of a western blot analysis using an anti-6x-His tag antibody or by visual inspection of the plants, as at least six of these effectors triggered cell death in the agroinfiltrated tissue. We are currently completing the cloning of the 35 effectors in a second binary vector and we soon test their transient expression in our select grapevine lines as well.


Climate change is expected to increase irrigation demand at the same time as reducing irrigation supply, and developing plant material with lower irrigation needs is crucial to adapt the grape industry to future conditions.  Previous work suggests that altering stomatal traits to reduce maximum gas exchange rates or reduce gas exchange more strongly in response to water stress would increase grapevine water-use efficiency without compromising the carbon supply for growth and ripening.  The goal of our project is to use genetic engineering to generate plant material with water-saving stomatal traits, while leaving the rest of the genome intact, to experimentally test the effects of these traits on yield and ripening in economically important grape cultivars.  We will target four genes with known effects on stomatal traits and gas exchange for over-expression (OST1 and CLE25) or under-expression (knock-out mutations) (EPFL9 and EVE).  We will conduct these transformations in Thompson Seedless, as a table grape with a high success rate for genetic transformation, and Chardonnay, as the most-produced wine grape in California, including in hot, water-limited regions.  This year, the first year of a proposed five-year project, we completed the bioinformatics work needed for the plant transformations.  The Cantu lab completed sequencing for the Thompson Seedless genome and identified homologues for the four target genes in the two grape varieties.  The Cantu lab is currently creating the plasmids containing the target mutations, which will be supplied to the UC Davis Plant Transformation facility to create the transformed plant material.

Furthering Elite Grape Rootstocks With Reduced Nematode Susceptibility

The long-time plantings of vineyards are continuously at risk for infection by soil-borne pests and diseases.  These have ample time and opportunity to establish, build-up in population densities and cause damage.  Depending on the severity of infection, plant-parasitic nematodes can become major yield constraints.  In vineyard soils, soil-dwelling plant-parasitic nematodes can reproduce on susceptible plant roots.  Multiple species are often found at a location.   Meloidogyne spp.  (root-knot nematodes), Mescocriconema xenoplax (ring nematode), Xiphinema americanum or X.  index (dagger nematode), and Tylenchulus semipenetrans (citrus nematode) are most frequent and most damaging.  These can occur in various combinations within at least the upper 5-ft soil layers.  Utility of resistant rootstocks is challenged under these conditions of multiple species being present.  In a prior project, rootstock genotypes that had been selected under greenhouse conditions were tested under field conditions in nematode-infested soils following a >30-year-old planting of ‘Thompson seedless’ grape.  They were grafted to ‘Pinot Gris’ and produced yields in 2022 and 2023.  Twelve genotypes were selected from these studies based on their propagation characteristics, vigor under nematode-infested conditions, and productivity of grape ‘Pinot Gris’.  In the current project year, dormant wood cuttings were made, bench-grafted to ‘Pinot Gris’, and planted to an experimental vineyard in early summer 2023.  Cleanliness from virus (GFLV, GLRaV-3, GRBV) of these cuttings was attested by Plant Foundation Services (FPS) before propagation.

Developing an Efficient DNA-free, Non-transgenic Genome Editing Methodology in Grapevine

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.

Evaluating Grape Rootstocks for Nematode Susceptibility

In California grape production is one of the perennial high-value commodities. Because of the traditional long time period a vineyard is cultivated, soil-borne pests and diseases have ample time and opportunity to build-up and be major yield constraints. Once introduced in vineyard soil, these soil-dwelling parasites can reproduce on susceptible plant roots copiously. Typically multiple species including Meloidogyne spp. (root-knot nematodes), Mescocriconema xenoplax (ring nematode), Xiphinema americanum or X. index (dagger nematode), and Tylenchulus semipenetrans (citrus nematode) and others occur in various combinations in vineyard soil and damage the crop. Once they are established in an existing vineyard they are difficult to manage because they colonize at least the upper 5-ft soil layers. Genetic resistance in rootstocks can offer protection from these culprits, and offer sustainable and efficient protection of the risk of nematode damage. The frequent co-occurrence of multiple plant-parasitic nematode species makes attaining highest levels of resistance against such nematode assemblies tedious. Typically, the resistance towards one species has limited to no effect on a different species. In the current project, selections of grape rootstock genotypes selected foremost under greenhouse conditions were exposed to field populations of nematode assemblies. In 2019, one experimental vineyard was planted following a >30 year old planting of Thompson seedless grape. A total of seventeen experimental rootstocks developed in Dr. Andrew Walkers program were planted alongside repeat entries from prior experimentation and longer-tested GRN1, GRN2, GRN4 (A. Walker) and 1017A. In addition, Zinfandel, St. George, Harmony, Flame, and Salt Creek were planted as commercial controls. In mid-June 2019, rootstocks were planted in randomized complete block design with five replications into this nematode-infested site. A month after planting, each planting site receive additional root-knot nematode-infested soil to boost the infestation levels. In 2020, another nine experimental rootstocks with similar controls were planted, and additionally inoculated with infested soil. First nematode assessments were done in the dormant season. Field testing these rootstock candidates will provide important information on the nematological and horticultural status of them. These activities will allow to identify elites that warrant further characterization, and that could add new so urgently needed rootstocks for the industry.


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.

Evaluating Traits To Improve Grapevine Water-Use Efficiency and Drought Tolerance

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 has 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 and growth in leaves and roots. A more negative TLP indicates the leaves and roots can undergo more severe water stress before losing turgor. 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 crops, but these traits have never been assessed for their impact on grapevine performance under water stress.

Our first year, we conducted a greenhouse experiment to evaluate whether rootstocks with a more negative root TLPdry and a larger DTLP allowed vines to maintain greater gas exchange, growth, and WUE under water stress. This year, we have reanalyzed these data and found significant correlations between a more negative TLPdry and larger root system, and between greater vine gas exchange under drought and lower values of a second trait, the root capacitance (CAPdry). CAP measures the volume of water lost from the root as the root dehydrates to TLP. A lower CAP indicates the root loses less volume, with potentially important effects on belowground water transport and root-to-shoot chemical signaling. However, TLPdry was significantly less negative in the rootstocks that field trials have classified as drought tolerant, suggesting that a less negative TLP in dry roots could benefit drought tolerance by redirecting resources to increase root growth in deeper, wetter soil. Together, these findings suggest that root TLP and CAP are promising traits to improve rootstock drought tolerance, though more work is needed to clarify their function.

Our second year, we used a vineyard experiment with 7 wine grape cultivars to test the impacts of leaf TLP and DTLP, and the biochemistry underlying variation in these traits, on scion drought tolerance, including gas exchange, vigor, and berry yield and quality, in realistic vineyard conditions. All 7 cultivars significantly adjusted TLP to improve leaf drought tolerance over the growing season, but cultivar rankings in TLP were highly consistent. However, contrary to expectation, leaf TLP was not related to gas exchange or plant water stress in this study, suggesting leaf biochemistry was responding to other stresses in these vines. Further, this study is the first to report a significant accumulation in mannitol as part of DTLP in wine grapes, which has been shown to ameliorate heat and pathogen stress in other plant species.

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’.

Development of next generation rootstocks for California vineyards

Since my last report (June 2019) Nina Romero has made excellent improvements to
our rootstock screening and is currently re-vamping our ring nematode resistance screening, and she has replaced three technicians who departed over the past year. There are 444 genotypes in testing for resistance to nematodes, salt or both. Our 2019 crosses again focused on using fertile and tetraploid VR hybrids to get rotundifolia forms of resistance into better rooting backgrounds, and mostly failed (due to the genetic distance between Vitis grape species and rotundifolia. We have been successful with crosses to two VR hybrids both of which resist phylloxera and combined with rootstocks. Seed from the 2018 crosses are mostly in storage for the next grape breeder, except for the 18113 (GRZN3 x V. acerifolia 9018. Chris Chen is working on his PhD with this population which brings excellent and broad nematode
resistance to our best form of salt tolerance (which also has strong root-knot resistance). We have improved our phylloxera screening in the greenhouse and have verified a number of fertile VR hybrids also have strong phylloxera resistance. A new post-doc (Erin Galarneau from the Baumgartner lab) was hired to direct examinations of phenolic compounds responsible for phylloxera and nematode resistance. They will also assist our efforts to determine how O39-16 induces fanleaf degeneration resistance. We are also making good progress on identifying the basis of red leaf virus tolerance. These efforts are being directed by a visiting scholar from China. We have rootstock examples of strong tolerance (St. George and AXR1) and very sensitive (Freedom and 101-14) and rapid tissue-culture and greenhouse-based
screens that are rapid and mimic field tests.

Evaluation of Grapevine Rootstock Selections

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. The initial plantings from the USDA rootstock breeding program number over 700 selections. This initial group was grown until maturity, and then evaluated for their ability to be good mother vines. This evaluation identified nearly 150 selections that were good to moderate mother vines. Selections with good mother vines qualities were tested by Dr. Andreas Westphal and Dr. Andrew Walker for resistance to aggressive root-knot nematodes. Selections that performed well in both assessments have been grafted to scions for in field evaluations. Currently six selections (PC0333-5, PC0349-11, PC0349-30, PC04153-4, PC0495-51, and PC0597-13) have been planted in a replicated trial in a commercial winegrape vineyard in Merced. These vines should start production during the 2020 growing season. Five selections had been grafted to a table grape and planted in a replicated trial in a commercial table grape vineyard in Delano, CA. Unfortunately, this trial has run into problems and only limited data will be collected. 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. Since 2016 a total of 165 new selections have been planted. These vines will need to undergo the same testing as the previous rootstocks once they are mature. It is hoped that a new table grape vineyard can be established with these advanced selections. The Merced trial 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 was completed during the 2019 growing season.