Source: RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY submitted to
IMPROVEMENT OF CROP DISEASE RESISTANCE AND STRESS TOLERANCE BY CRISPR/CAS GENE EDITING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1005372
Grant No.
(N/A)
Project No.
NJ12122
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Dec 1, 2014
Project End Date
Nov 30, 2019
Grant Year
(N/A)
Project Director
Di, RO, .
Recipient Organization
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
3 RUTGERS PLZA
NEW BRUNSWICK,NJ 08901-8559
Performing Department
Plant Biology & Pathology
Non Technical Summary
For the past few decades, molecular techniques and plant biotechnology have greatly aided our understanding of plant biology and have created crop plants with valuable new agronomic and nutritional traits for the benefits of farmers, consumers and the environment. Plants with stress tolerance, disease and pest resistance, herbicide resistance and superior nutritional composition have been produced through the approaches of transgene integration and RNAi (RNA interference)-gene silencing. However, transgenic plants or genetically modified organisms (GMOs) are mostly produced with a single transgene inserted into the plant genome, and the transgenes are often from heterologous organisms. It is difficult to engineer plants that are resistant to more than one pest through the single transgene integration. Engineering plants that are tolerant to a single environmental stress such as heat or drought is also a challenge, as complex reactions are elicited from plants under stress. Additionally, the integration of transgenes is random in the genome and hard to control. Technologies other than transgene integration should be explored to better engineer stress tolerance and pest resistance in plants. Increasing public concern regarding GMOs should also be addressed. Whole genome information and functional genomics have greatly enhanced our ability to engineer plant genomes in the past decade. As more information on the molecular mechanisms between plant and pest/stress interactions becomes available, it is feasible to manipulate plant genomes by disrupting the host factors that contribute to the pest and stress interactions. RNAi (RNA interference) technology has been used to produce plants that are resistant to diseases. However, RNAi normally leads to down-regulation rather than complete inhibition of target genes.The new, exciting and amazing gene editing technology termed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system that came into the spotlight about two years ago can potentially solve the problems encountered by conventional transgene and RNAi technologies. We propose to construct two CRISPR/Cas vectors for dicotyledonous and monocotyledonous plants (dicot and monocot) by targeting the negative regulators of disease resistance and stress tolerance. It is intended that these two vectors be used for any dicot or monocot plant, with at most the substitution of promoters for specific plant species. These two vectors are different from all the other CRISPR/Cas vectors used in plant gene editing: the 20-bp target sequence can be easily and efficiently substituted by other 20-bp sequences of any gene from any dicot or monocot plant with the simple Q5 site-directed mutagenesis kit (New England BioLabs) by an one-step PCR (polymerase chain reaction). Grape will be the dicot plant to be engineered for downy mildew disease resistance. Grape is an important commodity worldwide in the fruit market and wine industry. In New Jersey, the acreage of wine grapes doubled from 2002 to 2007 (Rutgers NJAES information). Currently, there are more than 190 farms statewide, in three designated American Viticulture Areas (AVAs). However, grapevine downy mildew caused by Plasmopara viticola is a serious disease affecting mainly Vitis vinifera varieties in New Jersey, as well as around the world. Cross-breeding and fungicide spray have been the main control measures for grapevine downy mildew disease for centuries. This new grape variety will require less fungicide applications, benefiting grape growers, consumers and the environment.Turfgrass will be the monocot to be engineered for dollar spot disease resistance and stress tolerance. Creeping bentgrass (Agrostis stolonifera L.) is one of the most widely used cool-season grass species on golf courses, bowling greens and tennis courts. However, there is no cultivar of creeping bentgrass that is considered to be completely resistant to dollar spot disease caused by Sclerotinia homoeocarpa F.T. Bennet. More than 70% of fungicide used on golf courses are to control dollar spot, brown patch (Rhizoctonia solani Kuhn), and anthracnose [Colletotrichum graminicola (Ces.) G.W. Wils]. Additionally, creeping bentgrass is stressed by heat and drought as other grass species during summer months. As turfgrass is a multi-billion dollar industry contributing to the national economy, disease resistant and stress tolerant turfgrass will benefit turfgrass industry and the environment.
Animal Health Component
90%
Research Effort Categories
Basic
10%
Applied
90%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121139104050%
2011629116050%
Goals / Objectives
The goal of this project is to develop the platform of CRISPR/Cas-based gene editing for both dicot and monocot plants to engineer disease resistance and stress tolerance using grape and turfgrass as model plants, both of which are important crops for New Jersey and the U.S. CRISPR/Cas gene editing is the new technology that can greatly enhance crop breeding programs to boost the agriculture production. The specific objectives are: (1)construct pDi-sgRNA vector for dicotyledonous plant gene targeting (we already have the pMo-sgRNA vecctor for monocotyledonouse plant gene targeting); (2)construct grape gene targeting vectors for the following three genes to engineer grape downy mildew disease resistance: flavanone 3-dioxygenase-like (VvF3DOL, XM_002273604.2), homoserine kinase-like (VvHSKL,XM_002277851.2) and the putative leucine-rich repeat receptor-like protein kinase-like (VvLRR-RLKL, XM_002276944.1); (3) clone creeping bentgrass CPK12, CBF2/DREB1C and nudC gene homolog genes; (4) construct creeping bentgrass gene targeting vectors for the following three genes to engineer turfgrass stress tolerance and disease resistance: CPK12,CBF2/DREB1C and nudC homologs; (5) transform grape and creeping bentgrass with the gene targeting vectors by gene gunbombardment; (6) screen for gene-targeted grape and creeping bentgrass mutant plants; and (7) testgene-edited grape and creeping bentgrassplants for disease resistance and stress tolerance.
Project Methods
1. Construct pDi-sgRNA vector for dicotyledonous plant gene targeting: The pMo-sgRNA vector has been made to target monocot plant genes with the following components: the rice miRNA U6 promoter, the 20-bp gene target sequence (of the first selected turfgrass CPK12 gene homolog) and the sgRNA scaffold, the maize ubiquitin promoter with an intron driving the monocot-optimized Cas gene followed by maize ubiquitin terminator. The rice miRNA U6 promoter will be substituted with the turfgrass miRNA U6 promoter when its sequence is known. The pDi-sgRNA vector will be similarly designed and synthesized. It will contain the Arabidopsis miRNA U6 promoter, the first selected grape VvF3DOL gene 20-bp target sequence, the sgRNA scaffold, the Arabidopsis ubiquitin promoter driving the dicot-optimized Cas gene followed by Arabidopsis ubiquitin terminator. The Arabidopsis miRNA U6 promoter will be substituted with the grape miRNA U6 promoter when its sequence is known.2. Construct grape gene targeting vectors for the following three genes to engineer grape downy mildew disease resistance: flavanone 3-dioxygenase-like (VvF3DOL, XM_002273604.2), homoserine kinase-like (VvHSKL, XM_002277851.2) and the putative leucine-rich repeat receptor-like protein kinase-like (VvLRR-RLKL, XM_002276944.1) The 20-bp gene target sequences conforming to the GN19NGG requirement will be chosen where N is any base, the initial G is the requirement for the initiation of U6 promoter, and the NGG is the PAM required for the Cas enzymatic activity.The GN19 is the 20-bp that will be driven by the U6 promoter and match to the genomic target site.All the target sites include a restriction enzyme site within the sequence to facilitate future screening of gene edited plants. The pDi-sgRNA vector will have the GN19 sequence built in for the VvF3DOL gene. Two sets of bi-directional oligonucleotides will be designed for the second grape gene VvHSKL, and the third grape gene VvLRR-RLKL. These sets of oligonucleotides will be used in PCR reactions with the Q5 site-directed mutagenesis kit from New England BioLabs to substitute the VvF3DOL GN19 sequence. These three VvsgRNA/Cas cassettes will be subcloned into the plant expression vector pCAMBIA1300 (www.cambia.org) with hygromycin resistance selectable marker.3. Clone creeping bentgrass (Crenshaw cultivar) CPK12, CBF2/DREB1C and nudC gene homologs: The peptide sequences of these three genes from rice and Brachypodium were aligned. The comparisons show that they share 80%, 51% and 72% homology over 541, 373 and 308 amino acids respectively. Exact primers for CPK12 homolog based on the cDNA clone sequence, and degenerative primers for the other two homologs will be designed for RT-PCR cloning. The cloned gene fragments will be sequenced and provide several 20-bp target sites for CRISPR/Cas-mediated gene editing.4. Construct creeping bentgrass gene targeting vectors for CPK12, CBF2/DREB1C and nudC homologs: The 20-bp gene target sequences conforming to the GN19NGG requirement will be chosen. The bentgrass gene targeting constructs will be first made in pMo-sgRNA vector and then subcloned into pCAMBIA1300 similarly to the construction of grape gene editing vectors.5. Transform grape and creeping bentgrass with gene targeting vectors by gene gun bombardment: We have obtained grape Vitis vinifera Chardonnay somatic embryogenic cultures from the USDA-ARS Grape Genetics Research Unit, New York State Agricultural Experiment Station. We have also started generating somatic embryogenic cultures for the New Jersey Chardonnay cultivar Dijon Clone 76. We will follow the well-developed grape and turfgrass transformation methods with modifications, the tissues will be transformed by the gene gun bombardment.6. Screen for gene-targeted grape and creeping bentgrass mutant plants: The integration of the sgRNA/Cas cassettes will be examined on T0 plants by PCR. Primers will be designed for the gene sequences spanning the mutation sites. PCR reactions will be conducted for all the regenerated plantlets, and PCR products digested with restriction enzymes. Since restriction enzyme sites are included in the mutagenesis sites, no digestion of any PCR product will indicate a gene-edited line of grape and creeping bentgrass. The undigested PCR fragments will be sequenced to confirm the mutations. Gene-edited grape and Crenshaw plants will be clonally propagated.7. Test gene edited grape and creeping bentgrass plants for disease resistance and stress tolerance:Gene edited grape will be tested for resistance to P. viticola by the leaf disc assay.The pathogen will be collected from New Jersey and maintained on grape leaf discs at -20 oC and sub-cultured. Sporangia will be collected, suspended in sterile water, inoculated onto the leaf discs of wild type and gene-edited grape plants. The leaf discs in petri dishes with water agar will be incubated at 22 oC with a photoperiod of 16/8 hr (light/dark). Disease severity will be scored. To test the gene-edited Crenshaw plants for drought tolerance, watering will be withheld for 15 days from whole plants growing in pots under greenhouse conditions.Whole plants will be tested for cold tolerance by exposing them to 4 oC for 6 days.Whole plants will also be tested for salinity tolerance by watering with 100 mM NaCl for 2 days followed by 200 mM and 300 mM NaCl for 2 days each or as needed.Plants will then be returned to normal conditions. Gene-edited plants' performance and survival rate will be recorded and compared to wild type clonally propagated Crenshaw plants.Proline and soluble sugar levels in plants will be measured after withholding water, cold or salt-treatment for 24 hr.Leaves from stressed plants will be quick-frozen in liquid nitrogen and homogenized.The homogenates will be extracted with 80% methanol and boiled for 10 min.The proline and soluble sugar contents will be analyzed by high performance liquid chromatography. To test the gene-edited Crenshaw plants for S. homoeocarpa resistance, both detached leaf assay and whole plant inoculation will be performed. For the detached leaf assay, the second expanded leaves from the top of plant stolons will be used. Leaves will be disinfected with 70% ethanol, rinsed with sterile water, placed on 1% agar in Petri dishes and infected with S. homoeocarpa mycelium grown on potato dextrose agar at 25 oC for 3 days. Petri dishes will be put under 14/10 hr (day/night) photoperiod at 28 oC and 70% humidity. The development of dollar spot disease will be rated by measuring the lesion length 2, 4, 7 days post inoculation. Plants grown in pots will be directly inoculated with rye seeds colonized by S. homoeocarpa, and placed in plastic trays in greenhouse with 14/10 hr (day/night) photoperiod and 22 oC/light and 17 oC/dark. Disease severity will be visually assessed at 3, 5, 7 and 9 days post inoculation.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One of my graduate students, Yee Chen Low, who is supported by our Teaching Assistantship, worked on the cloning of the grape downy mildew susceptibility genes, making constructs for Arabidopsis and grape transformation, transforming Arabidopsis and analyzing the Arabidopsis gene-edited mutants. Additionally, this project has provided the hand-on research experience of gene cloning and plant genetic engineering for several undergraduate students in the majors of plant biology and biotechnology at Rutgers University. 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?Goal #1: Accomplished. Goal #2: Grape gene editing vectors have been constructed. Downy mildew (DM) susceptibility gene-edited Arabidopsis mutant plants have been produced. We will characterize the DM resistance of these gene-edited Arabidopsis mutants. We will also transform the gene-edited Arabidopsis mutant plants with the grape VvF3DOL and VvHSK genes to validate their involvement in the grape susceptibility to DM pathogen. Goal #3: Accomplished. Goal #4: As we have cloned the partial cDNA and gDNA sequences of the AsBON1, AsCPK12 andAsDREB1C genes, we will construct the CRISPR-editing vectors to be used to knock these genes out in Crenshaw creeping bentgrass. Goal #5: We will continue to screen pRD220-, pRD235- and pRD236-bombarded NJ76 calli and regenerate transformed plantlets and characterize the gene mutations in the regenerants. When the CRISPR vectors are ready for the AsBON1, AsCPK12 andAsDREB1C gene, they will be used to transform creeping bentgrass calli. Goal #6 and Goal #7: When gene-targeted grape and creeping bentgrass mutant plants are obtained, we will characterize the gene mutations by RFLP (restriction fragment length polymorphism) and T7E1 endonuclease analysis. We will later testthe gene-edited grape and creeping bentgrassplants for disease resistance and stress tolerance.

Impacts
What was accomplished under these goals? CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) gene editing is the new technology that can greatly enhance crop breeding programs to boost the agriculture production. The goal of this project is to develop the platform of CRISPR/Cas-based gene editing for both dicot and monocot plants to engineer disease resistance and stress tolerance using grape and turfgrass as model plants, both of which are important crops for New Jersey and the U.S. It is expected that gene edited grape plants will have the downy mildew susceptible genes knocked out. This new grape variety will require less fungicide applications, benefiting grape growers, consumers and the environment. For turfgrass, CRISPR/Cas gene editing technology will greatly enhance the breeding program at our world-class Rutgers Center for Turfgrass Science to produce disease resistant and stress tolerant turfgrass germplasm. As turfgrass is a multi-billion dollar industry contributing to the national economy, disease resistant and stress tolerant turfgrass will benefit turfgrass industry and the environment. Goal #1: Construct CRISPR vectors for monocotyledonous and dicotyledonous plant gene targeting. We have constructed the CRISPR vector for grass plant gene targeting, pRD213, which includes the wheat U6 miRNA promoter driving the 20-bp target sequence and the crRNA scaffold (together as guide RNA or gRNA), and the Cas9 nuclease with monocot-specific codons driven by the maize ubiquitin promoter and terminated by the nopaline synthase (NOS) 3' sequence. pRD213 is in the backbone of pGEM3Zf(+) cloning vector, which allows the insertion of the 20-bp target sequence of any chosen monocot gene, and the subcloning of the gRNA/Cas9 cassette into the pCAMBIA1300 plant expression vector. For grape gene editing, we have adopted a new strategy to construct CRISPR gene editing vectors by utilizing two flanking ribozymes to express the 20-bp target sequence and the crRNA scaffold (gRNA). We have constructed the vector, pRD216, containing the Arabidopsis ubiquitin promoter driving the ribozymes-flanked grape homoserine kinase gene (VvHSK) 20-bp target sequence and the crRNA scaffold, and the humanized Cas9 nuclease gene driven by the 2× CaMV 35S promoter and terminated by the NOS terminator. pRD216 can be used to swap the 20-bp target sequence of any chosen dicot genes. Goal #2: Construct grape (Vitis vinifera, Vv) gene targeting vectors for the following two genes to engineer grape downy mildew disease resistance: flavanone 3-dioxygenase-like (VvF3DOL, XM_002273604.2) and homoserine kinase (VvHSK,XM_002277851.2). The plasmid pRD216 mentioned above is the intermediate vector containing the grape HSK target sequence. The VvHSK gRNA/Cas9 cassette has been subcloned into plant expression vector pCAMBIA2300, resulting in pRD220 with kanamycin selectable marker. As a backup plan, we have also constructed the VvHSK- and VvF3DOL-CRISPR vectors, pRD235 and pRD236, with the Arabidopsis U6 promoter driving the gRNAs. Since the relationship between the putative VvHSK and VvF3DOL genes and grape downy mildew (DM) resistance is largely unknown, we have decided to use Arabidopsis thaliana (At) and its DM pathogen Hyaloperonospora parasitica as a model to study the interaction between plant and DM pathogens. We have since constructed the following two CRISPR plasmids, pRD207 and pRD212, to knock-out the 2OG-Fe(II) oxygenase (At2OGFeO, homolog to VvF3DOL) and the AtHSK (homolog to VvHSKL) genes in Arabidopsis. The gRNAs in these two plasmids are driven by the Arabidopsis U6 miRNA promoter. As the cDNA insertion null mutants of Arabidopsis 2OG-Fe(II) oxygenase and HSK demonstrate significantly improved resistance to downy mildew, we anticipate that our CRISPR-edited Arabidopsis mutants will display similar resistance. We have produced several At2OGFeO (pRD207) and AtHSK (pRD212) deletion mutants. Furthermore, we have selected transgene (gRNA+Cas9)-free At2OGFeO (pRD207) T2 mutants. Initial testing on the resistance of pRD207 T2 plants with H. parasitica Noco2 strain inoculation and RT-qPCR quantification of the pathogen showed that these At2OGFeO (pRD207) T2 mutants had much lower pathogen level than the wild type Arabidopsis Col-0 plants. We then will carry out the "susceptibility rescue" experiment with the grape VvF3DOL and VvHSK cDNAs in the CRISPR-edited Arabidopsis mutants. If the DM susceptibility is restored in the CRISPR-editing Arabidopsis mutant plants to H. parasitica, we will be ascertained that the VvF3DOL and VvHSK genes that we identified are indeed involved in the grape susceptibility to grape DM pathogen. We have cloned the VvHSKL and VvF3DOL genes from the NJ76 Chardonnay cultivar by RT-PCR. The cDNA of these two genes have been cloned into a general plant expression vector, resulting in pRD240 and pRD241, which will be used to transform into the CRISPR-edited Arabidopsis mutant plants. Goal #3: Clone creeping bentgrass (Agrostis stolonifera L., As) BON1, CPK12 and DREB1C homolog genes. We originally intended to work with nudC gene in creeping bentgrass. This gene was found to be difficult to clone. Instead, we found out that the BON1 (BONZAI1) gene is a repressor of disease resistance gene in Arabidopsis, and BON1 null mutant was shown to display enhanced resistance to disease. Therefore, our research has been focused on the AsBON1, AsCPK12 and AsDREB1C genes. Using bioinformatics analysis and the existing partial EST (expressed sequence tags) library of creeping bentgrass Crenshaw cultivar, we have cloned partial cDNAs for these three genes. We have also cloned partial genomic DNA sequences of these three genes from Crenshaw. Goal #4: Construct creeping bentgrass gene targeting vectors for the following three genes to engineer turfgrass stress tolerance and disease resistance: AsBON1, AsCPK12 andAsDREB1C. As we have obtained the partial genomic DNA sequences of these three creeping bentgrass genes, we have identified target sequences with convenient restriction enzymatic sites. We have swapped the target sequence in pRD213 with the target sequences of AsBON1, AsCPK12 andAsDREB1C by mutagenesis. The CRISPR-gene editing vectors for the AsBON1, AsCPK12 and AsDREB1C genes are being constructed with the gRNAs driven by the wheat U6 promoter and the monocot codon-optimized Cas9 gene under the control of the maize ubiquitin promoter. Goal #5: Transform grape and creeping bentgrass with the gene targeting vectors by gene gunbombardment. We have initiated embryogenic calli culture from the grapevine buds of New Jersey Chardonnay cultivar NJ76. We have developed the tissue culture protocol to regenerate grape plantlets from the embryogenic calli. We have also developed tissue culture protocol for creeping bentgrass from mature seed-derived calli to regenerated plants. pRD220 containing the VvHSK gRNA/Cas9 cassette with the ribozyme design and kanamycin selectable marker has been bombarded into NJ76 calli. The pRD220-transformed NJ76 calli have been selected on G418-containg medium. The expression of Cas9 has been shown by Western blot analysis with Cas9-specific antibody. We are in the process of regenerating pRD220-transformed grape plants. We have also transformed newly initiated NJ76 embryogenic calli with the constructs of pRD235 and pRD236 by gene gun bombardment.

Publications


    Progress 12/01/14 to 09/30/15

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Dr. Haekeun Yun from the Department of Horticultural Bio-Science, Yeungnam University, Korea, was supported by his university, worked on the grape goals from Sept. 1, 2014 to Aug. 31, 2015. Dr. Yun's visit and work expanded our collaboration to other institutes in Korea. This project has trained Dr. Yun, an established grape breeder, in molecular breeding of crops. One of my graduate students, Yee Chen Low, who is supported by our Teaching Assistantship, volunteered her time in cloning the grape downy mildew susceptibility genes and making constructs for Arabidopsis and grape transformation. Additionally, two of my undergraduate students, Kuan Yu Cheong and Richard Darryl Dominic, participated in the cloning of turfgrass stress-related genes and turfgrass tissue culture. This project has provided the hand-on research experience of gene cloning and plant genetic engineering for both Kuan Yu and Darryl. 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?Goal #1: Accomplished. Goal #2: We will construct the CRISPR vector containing the VvHSKL sgRNA/Cas9 cassette for grape transformation. Goal #3: Since we have obtained partial 150-250 bp gDNA and cDNA clones of the As CPK12,CBF2/DREB1C and nudC homolog genes, we will carry out the inverse PCR reactions to clone longer As gDNA fragments. Goal #4: When 400-500 bp gDNA fragments are obtained for As CPK12,CBF2/DREB1C and nudC homolog genes, CRISPR vectors will be constructed to include the sgRNA sequences targeting to these three As genes. Goal #5: We will continue to screen pRD220 (VvHSKL sgRNA/Cas9)-bombarded NJ76 calli and regenerate transformed plantlets and characterize the gene mutations in the regenerants. We will transform NJ76 calli with the construct containing VvF3DOL sgRNA/Cas9 when the plasmid is made. We will characterize the At 2OG-Fe(II) oxygenase and At HSK gene mutations in the CRISPR-edited Arabidopsis plants. When gene-edited Arabidopsis plants are confirmed, they will be transformed with the pRD240 and pRD241 constructs containing the VvHSKL and VvF3DOL cDNAs to perform the "susceptibility rescue" experiments to validate the involvement of the cloned VvHSKL and VvF3DOL genes in the interaction of grapevine and downy mildew pathogen. When the CRISPR vectors are ready for the As CPK12,CBF2/DREB1C and nudC homolog genes, they will be used to transform creeping bentgrass calli. Goal #6 and Goal #7: When gene-targeted grape and creeping bentgrass mutant plants are obtained, we will characterize the gene mutations by RFLP (restriction fragment length polymorphism) and T7E1 endonuclease analysis. We will later testthe gene-edited grape and creeping bentgrassplants for disease resistance and stress tolerance.

    Impacts
    What was accomplished under these goals? CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) gene editing is the new technology that can greatly enhance crop breeding programs to boost the agriculture production. The goal of this project is to develop the platform of CRISPR/Cas-based gene editing for both dicot and monocot plants to engineer disease resistance and stress tolerance using grape and turfgrass as model plants, both of which are important crops for New Jersey and the U.S. It is expected that gene edited grape plants will have the downy mildew susceptible genes knocked out. This new grape variety will require less fungicide applications, benefiting grape growers, consumers and the environment. For turfgrass, CRISPR/Cas gene editing technology will greatly enhance the breeding program at our world-class Rutgers Center for Turfgrass Science to produce disease resistant and stress tolerant turfgrass germplasm. As turfgrass is a multi-billion dollar industry contributing to the national economy, disease resistant and stress tolerant turfgrass will benefit turfgrass industry and the environment. Goal #1: Construct CRISPR vector for dicotyledonous plant gene targeting. We have constructed the CRISPR vector for monocotyledonouse plant gene targeting, pRD213, which includes the wheat U6 miRNA promoter driving the 20-bp target sequence and the crRNA scaffold, and the Cas9 nuclease with monocot-specific codons driven by the maize ubiquitin promoter and terminated by the nopaline synthase (NOS) 3' sequence. pRD213 is in the backbone of pGEM3Zf(+) cloning vector, which allows the insertion of the 20-bp target sequence of any chosen monocot gene, and the subcloning of the sgRNA (single guide RNA)/Cas9 cassette into the pCAMBIA1300 plant expression vector. For gene editing grape, a dicot plant, the grape U6 miRNA promoter information is not available; however, we can use the CRISPR vector with the Arabidopsis U6 miRNA promoter. Since the start of this project, we have adopted a new strategy to construct CRISPR gene editing vectors by utilizing two flanking ribozymes to express the 20-bp target sequence and the crRNA scaffold (sgRNA), negating the necessity of the often-unavailable miRNA promoters. We have constructed the vector, pRD216, containing the Arabidopsis ubiquitin promoter driving the ribozymes-flanked grape HSKL (homoserine kinase-like) 20-bp target sequence and the crRNA scaffold, and the humanized Cas9 nuclease gene driven by the 2× CaMV 35S promoter and terminated by the NOS terminator. pRD216 can be used to swap the 20-bp target sequence of any chosen dicot genes. Goal #2: Construct grape (Vitis vinifera, Vv) gene targeting vectors for the following three genes to engineer grape downy mildew disease resistance: flavanone 3-dioxygenase-like (VvF3DOL, XM_002273604.2), homoserine kinase-like (VvHSKL,XM_002277851.2) and the putative leucine-rich repeat receptor-like protein kinase-like (VvLRR-RLKL, XM_002276944.1). From publications and communications with other scientists, we have learned that there are hundreds of LRR-RLKL genes in grape, it will be not very meaningful to knock-out a single grape LRR-RLKL gene, so we have decided to focus on the VvF3DOL and VvHSKL genes. The plasmid pRD216 mentioned above is the intermediate vector containing the grape HSKL target sequence. The VvHSKL sgRNA/Cas9 cassette has been subcloned into plant expression vector pCAMBIA2300, resulting in pRD220 with kanamycin selectable marker, which is ready to transform grape. After we started the project, we realized that Arabidopsis thaliana (At) and Hyaloperonospora parasitica have been used as a model to study the interaction between plant and downy mildew. We have since constructed the following two CRISPR plasmids, pRD207 and pRD212, to knock-out the 2OG-Fe(II) oxygenase (homolog to VvF3DOL) and the HSK (homolog to VvHSKL) genes in Arabidopsis. The sgRNAs in these two plasmids are driven by the Arabidopsis U6 miRNA promoter. As the cDNA insertion null mutants of Arabidopsis 2OG-Fe(II) oxygenase and HSK demonstrate significantly improved resistance to downy mildew, we anticipate that our CRISPR-edited Arabidopsis mutants will display similar resistance. We then will carry out the "susceptibility rescue" experiment with the grape VvF3DOL and VvHSKL cDNAs in the CRISPR-edited Arabidopsis mutants. If the downy mildew susceptibility is restored in the CRISPR-editing Arabidopsis mutant plants to H. parasitica, we will be ascertained that the VvF3DOL and VvHSKL genes that we identified are indeed involved in the grape susceptibility to grape downy mildew pathogen. We have cloned the VvHSKL and VvF3DOL genes from the NJ76 Chardonnay cultivar by RT-PCR. The cDNA of these two genes have been cloned into a general plant expression vector, resulting in pRD240 and pRD241, which will be used to transform into the CRISPR-edited Arabidopsis mutant plants. Goal #3: Clone creeping bentgrass (Agrostis stolonifera L., As) CPK12, CBF2/DREB1C and nudC gene homolog genes. There is no complete genomic information for a single turfgrass species in GenBank. The EST (expressed sequence tags) library of creeping bentgrass Crenshaw cultivar that was constructed by Rutgers in 2007 was searched using the gene sequences of CBF2/DREB1C, CPK12 and LOC_Os06g12530 from rice (Oryza sativa) or Brachypodium distachyon as references by the tblstx and tblastn functions of GenBank website. The EST clone pDV865579 has been identified as a likely candidate for the creeping bentgrass partial CPK12 cDNA clone. Primers were designed based on the cDNA sequence of pDV865579, and used to clone the As CPK12 gDNA (genomic DNA) by PCR. Partial gDNA for As CPK12 has been obtained. Similarly, EST clones DY543604.1 and DV865150.1 have been identified from GenBank as potentially partial cDNA clones for As CBF2/DREB1C and As nudC-like genes. Primers have been designed and used to clone these two genes by RT-PCR. Partial cDNAs for these two genes have been obtained. Goal #4: Construct creeping bentgrass gene targeting vectors for the following three genes to engineer turfgrass stress tolerance and disease resistance: CPK12,CBF2/DREB1C and nudC homologs. This goal is in progress. Goal #5: Transform grape and creeping bentgrass with the gene targeting vectors by gene gunbombardment. We have initiated embryogenic calli culture from the grapevine buds of New Jersey Chardonnay cultivar NJ76. We have developed the tissue culture protocol to regenerate grape plantlets from the embryogenic calli. We have also developed tissue culture protocol for creeping bentgrass from mature seed-derived calli to regenerated plants. pRD220 containing the VvHSKL sgRNA/Cas9 cassette with the ribozyme design and kanamycin selectable marker has been bombarded into NJ76 calli. The pRD220-transformed NJ76 calli are being selected on G418-containg medium. pRD240 and pRD241 containing the sgRNA/Cas9 cassettes for At 2OG-Fe(II) oxygenase and At HSK genes have been used to Agrobacterium-transform Arabidopsis Columbia. Some transgenic Arabidopsis plants have been obtained on hygromycin-containing seed germination medium.

    Publications