IMPROVING GRAPEVINE RESISTANCE TO POWDERY MILDEW UTILIZING GENOME EDITING TECHNOLOGY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1026038
• Grant No.: 2021-38821-34513
• Cumulative Award Amt.: $499,999.00
• Proposal No.: 2020-11023
• Project Start Date: Apr 1, 2021
• Project End Date: Mar 31, 2025
• Grant Year: 2021
• Program Code: [EQ]- Research Project

Recipient Organization
University of Maryland Eastern Shore
11868 College Backborne Road
Princess Anne,MD 21853

Performing Department
Agriculture

Non Technical Summary
The goal of the project is to utilize techniques in precision breeding and genome editing for the development of powdery mildew resistant grape cultivars. Grapevine is affected by several fungal diseases, which are a serious threat to grape production worldwide. Among the fungal diseases affecting grapevine, powdery mildew is the most devastating disease worldwide. Specific objectives include 1) Identification and isolation of target MLO candidate genes from the Vitis genome for the development of CRISPR/Cas9 constructs, 2) Targeting MLO genes in embryogenic cultures to generate edited grape plant lines, 3) Greenhouse screening of edited plant lines to powdery mildew resistance and 4) Quantifying plant response to powdery mildew infection using scanning electron microscopy.The project utilizes grape genome information along with genome editing technology to generate targeted mutations while keeping desired viticultural traits intact. Genome editing, which involves modification of DNA sequences solely from the grape genome, is consumer and ecofriendly inasmuch it disrupts the genome much less than traditional breeding and should cause no GMO-related concerns. Disease resistant grape cultivars will provide major advantages such as decreased production costs, dramatically reducing the amount of fungicides thereby lowering exposure of workers and non-target organisms, and improvement of biodiversity in vineyards. The project will provide opportunities to minority graduate and undergraduate students thereby enhancing capacity building in research and teaching. Multi-disciplinary expertise of collaborators will complement individual programs and expand cutting edge research in areas of genomics and plant breeding that can be utilized by 1890 institutions for minority student research and training.

Animal Health Component

30%

Research Effort Categories

Basic

50%

Applied

30%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1139	1081	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1139 - Grapes, general/other;

Field Of Science
1081 - Breeding;

Keywords

disease resistance

genome editing

grapevine

powdery mildew

Goals / Objectives
The grapevine is among the most ancient crops cultivated by mankind.Grapevine cultivation is currently practiced in every state of the continental United States with CA, WA and NY accounting for more than 95% of the acreage in the United States. Grape is also a well-established crop in parts of the Eastern US, while acreage is rapidly expanding in other states including Maryland. Current grape acreage in MD stands at approximately 1000 acres with 89 licensed wineries statewide.Grapevine is affected by a number of fungal diseases, which are a serious threat to grape production worldwide. Management of grapevine diseases is accomplished using diverse strategies such as using certified clean planting material and chemical sprays for ensuring sustainable yields and high quality fruit.The goal of the current project is to utilize techniques in precision breeding and genome editing for the development of powdery mildew resistant grapevine cultivars. Specific project objectives include 1) Identification and isolation of target MLO candidate genes from the Vitis genome for the development of CRISPR/Cas9 constructs, 2) Targeting MLO genes in grape embryogenic cultures to generate edited plant lines of V. vinifera and Vitis hybrid cultivars, 3) greenhouse screening of edited plant lines for powdery mildew and 4) quantifying plant response to infection using scanning electron microscopy.

Project Methods
We hypothesize that the grapevine Mildew Locus O (MLO) and Resistance to Erisiphae necator (REN) gene families that are involved in powdery mildew infection process can be identified in different Vitis species and targeted to improve disease resistance using CRISPR/Cas9. The MLO and REN gene families will be screened in different Vitis species using the Pinot Noir reference genome and other online genomic databases to identify target genes for generating knockouts. Guide RNA's (gRNA) will be designed once unique sequences regions in target genes are identified. The gRNA sequences will be under the control of Arabidopsis U6-29 promoters and a Vitis aestivalis U6- 26 promoter for expression in cell cultures. Embryogenic cell cultures will be initiated from V. vinifera cultivars Thompson Seedless, Merlot and Seyval Blanc. Somatic embryos at the mid-cotyledonary stage of development will be used for targeting genes of interest. The CRISPR constructs and ribonucleoprotein complexes (RNP) and inserted in grape embryogenic cell cultures. Edited lines will be regenerated and acclimated prior to greenhouse transfer. The efficiency of editing in cell and plant lines will be recorded using quantitative-real time PCR. Whole genome sequencing will be carried out to study identify homozygous and heterozygous mutant lines, the effect of targeted mutations on plant phenotypes, and any unintended/off-target mutations and their effects on plant phenotypes. Edited lines will be transferred to a greenhouse and clonally propagated to make replicated clones. Edited lines will be screened in greenhouse tests for their response to Erisiphae necator infection along with non-transformed controls and a powdery mildew resistant control Vitis interspecific hybrid 'Tampa'. Similar studies will be carried out in laboratory assays using leaf discs from different sources to study the infection response. Resulting leaf discs will be used to study oxidative stress response and estimate the hyphal number on edited and control lines. The stained tissues will then be subjected to histology followed by scanning electron microscopy to determine the degree of fungal colonization and infection in the edited lines and controls. The colonization data will be correlated with greenhouse screening trials to identify lines showing varying degrees of powdery mildew resistance. Such resistant lines will then be subjected to molecular analyses to confirm the presence of any unintended transgene sequence insertion prior to them being utilized for further improvement and eventual commercialization (which will be beyond the scope of the current project).

Progress 04/01/23 to 03/31/24

Outputs
Target Audience:The target audience reached during this reporting period included undergraduate and graduate students and faculty, small and marginal farmers, and private companies involved in table grape breeding for disease resistance. Two research presentations were made at the biennial ARD symposium, which enabled the information to be delivered to students from other HBCU institutions. Additionally, research presentations were made at other conferences to disseminate the information to national and international audiences. Changes/Problems:We experienced some unexpected setbacks in the greenhouse screening process. In November 2023, the greenhouse complex at UMES lost heating capabilities for 6 weeks. This resulted in the death of some edited plant lines. The greenhouse propagation system was also rendered non-functional due to which clones from edited plant lines could not be produced. Additionally, the low temperatures in the greenhouse resulted in the death of the powdery mildew inoculum prevalent in the greenhouse and vines could not be screened following restoration of the heating system due to the absence of the mildew inoculum. Since plants could not sustain the powdery mildew infection, we were unable to carry out the histology and scanning electron microscopy work to quantify the infection process in the leaves of edited vines. A no-cost extension has enabled us to get back on track to complete the remaining activities for the project. What opportunities for training and professional development has the project provided?Undergraduate and graduate students were trained in plant tissue culture, genetic transformation and greenhouse screening of plant lines. How have the results been disseminated to communities of interest?The results were disseminated at the biennial Association of Research Directors (ARD) symposium, the Plant Biology 2024 conference and the2024 European Horticultural Congress. Additionally, a workshop on grape production and diseases affecting grapevines was conducted at the 2023 University of Maryland Eastern Shore, Small Farm Conference. What do you plan to do during the next reporting period to accomplish the goals?Leaf samples from edited and control lines that have been subjected to powdery mildew infection will be collected and analyzed for the estimation of hyphal density. Additionally, scanning electron microscopy will be used to study any difference in infection between control and edited vines.A detailed molecular analysis of the edited lines exhibiting resistance, including the identification of edited sites in the genome, off-target editing and enzymatic assays to determine the levels of peroxidase in edited and control vines will also be conducted.

Impacts
What was accomplished under these goals? A total of 105 independent edited lines were recovered following Agrobacterium-mediated transformation. Genomic DNA was isolated from independent plant lines and used for downstream applications. PCR analysis was conducted to determine the transgenic status of plants by using primers specific to the hygromycin selectable marker. A forward and a reverse primer spanning the entire region of the MLO-6 and MLO-7 genes were used to amplify the targeted region and identify any editing events in plant lines. PCR products were inserted into a cloning vector and sent for sequencing. Among the various plant lines tested, single nucluetodie insertions and deletions were detected in nine plant lines while a large portion of the MLO 6 gene was deleted in two plant lines. Independent plant lines were asexually propagated using cuttings to produce 3 replicated vines and screened to test their response to powdery mildew infection. Among the 47 plant lines, 5 exhibited varying levels of resistance to infection by powdery mildew compared to the non-edited control plants. We continue to conduct sequencing analyses and screen additional plant lines for their response to powdery mildew infection.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Dhekney, S.A., Sardaru, P., Jackson, C., and C. Wood. 2024. Applications of precision breeding and genome editing for Vitis trait improvement. Association of Research Directors (ARD) symposium, Nashville, TN, April 6-10.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: C.L. Jackson, C.Wood, P. Sardaru, A. Weaver and S.A. Dhekney. 2024. Evaluation of In Vitro Culture Response of Table Grape Cultivars for Vitis Genome Editing. P 700-119 Annual Conference of the American Society of Plant Biologists, Honolulu, HI June 22-26
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Sardaru, P., Dai, X., Zhao, Y and S.A. Dhekney Precision Breeding and Genome Editing of Wine and Table Grape Cultivars for Improving Disease Resistance. Acta Horticulturae, In Press

Progress 04/01/22 to 03/31/23

Outputs
Target Audience:The target audience included undergraduate and graduate students obtaining degrees in agriculture and food sciences, Maryland grape growersengaged in fruit and wine production and international researchers conducting research in viticulture. Changes/Problems:The growth of edited plant lines was hampered under greenhouse conditions due to a breakdown in heating for six weeks during the onset of winter. This slowed the progreess in the production of replicated clones and their screening for powdery mildew infection, and has delayed the screening proecess by one season. We hope to obtain results from greenhouse screening by Spring 2024 . What opportunities for training and professional development has the project provided?Undergraduate and graduatestudents were trained in areas of embryogenic culture establishment, genetic transformation and gene editing and the regeneration of edited plants. Students were trained in molecular biology areas including amplification of transgenes and edited sequences using PCR and the identification of insertions and deletions occuring from gene editing, in targeted areas of the grape genome. Students were also trained in writing manuscripts and abstracts for publications and making oral presentations at regional and national conferences. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Confirmation of gene editing in regenerated plant lines will be accomplished by molecular analysis incluidng PCR and genome sequencing. Plant lines will be screened under greenhouse conditions for their response to powdery mildew infection. Any correlations between editing and resistance will be identified. Additionally, scanning electron microscopy will be conducted to study any potential differences in quantifying hyphal density between edited lines and control plants.

Impacts
What was accomplished under these goals? Agrobacterium-mediated transformation with CRISPR/Cas9 constructs targeting the MLO 6 and MLO 7 candidate genes were used to produced edited plant lines in Vitis vinifera cultivars 'Thompson Seedless' and 'Bruce Sport'. A total of 69 embryo lines and 55 plant lines were recovered from both cultivars. Genomic DNA was isolated from independent plants lines. PCR analysis was conducted to determine the transgenic status of plants by using primers specific to the hygromycin selectable marker. Additionally, primers spanning the targeted portions of the MLO 6 and MLO 7 gene sequences were designed to determine the editing status of plants and the characterize insertions and deletions that might have occurred due to the editing process. Amplified PCR products were inserted into a cloning vector and sent for sequencing. We are currently awaiting sequencing results to determine the edited status of plants. Whole genome sequencing will be conducted for selected edited plant lines to determine the frequency of off-target editing. Regenerated plant lines were transferred to a greenhouse and are being multiplied using asexual propagation (cuttings). Four replicated clones from independent plant lines are currently being screened for their response to powdery mildew infection under greenhouse conditions.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Dhekney, S.A., Sardaru, P., Jackson, C., Fitts, J.M, Dai, X, and Zhao. Y. 2023. Genetic improvement of table and wine grape cultivars via precision breeding and genome editing technology. XIII International Conference on Grapevine Breeding, Genetics and Management. Cappadocia, Turkey, Aug. 21-24.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Sardaru, P., Jackson, C., Dai, X, Zhao. Y., and Dhekney, S.A. 2023. Optimizing CRISPR/Cas9-mediated genome editing of Vitis for trait improvement. Abstract # 800-163. Annual conference of the American Society of Plant Biologists, Savannah GA, Aug 5-9.

Progress 04/01/21 to 03/31/22

Outputs
Target Audience:Undergraduate and graduate students were trained in plant tissue culture and genome editing techniques. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Undergraduate and graduate students were trained in cell culture and genome editing techniques. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Edited plants will be produced in the next 12-14 weeks. We will conduct molecular analyses to determine the efficiency of editing, off-targets and biallelic mutations. Following molecular analyses, plants will be grown in a greenhouse and screened for their response to infection by powdery mildew.

Impacts
What was accomplished under these goals? The MLO 6 and MLO 7 gene sequences in Vitis were analyzed using online genomic databases to determine identify between the Thompson Seedless and Pinot Noirsequences. Following sequence analysis, we identified 2 guide RNA sequences that couldsimultaneously target the MLO6 and MLO 7 genes in grapevine. The guide RNA's were amplified using PCR and placed under the control of the Arabidopsis U26 and U29 promoters. The entire block was then placed in a binary vector containing Cas9 either under the control of a CaMV 35S or an embryo specific promoter. Two binary vectors were generated, which were transferred to EHA 105 and GV3101 using freeze thaw method of bacterial transformation. Embryogenic cultures of "Thompson Seedless", "Bruce Sport" were initiated from leaves of micrporpoagation cultures. Cultures were maintained in the dark for 9 weeks followed by transfer to light for 6 weeks. Resulting callus cultures were transferred to X6 medium for development and proliferation of somatic embryos. Following embryo proliferation for 6 weeks, they were used for co-cultivation with Agrobacterium harboring the CRISPR constructs. We are currently growing cultures transformed with the constructs on callus proliferation medium and expect to obtain edited embryos and plants within the next 12-14 weeks.

Publications