Progress 12/01/14 to 11/30/19
Outputs
Target Audience:Grape farmers and breeders. Turfgrass breeders. 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, 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 and other graduate students in the majors of plant biology and biotechnology at Rutgers University. How have the results been disseminated to communities of interest?Our results have been incorporated into the undergraduate and graduate courses that I teach at Rutgers University. A reporter has published an article about CRISPR gene editing in grape plants based on my information that have attracted some major grape growers. As a result, we have developed a cooperative project with a grape grower association. Acooperative project has also been developed with a turfgrass breeding company. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
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 (gRNA), and the Cas9 nuclease with monocot-specific codons driven by the maize ubiquitin promoter. For grape gene editing, 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 (the RGR design), and the humanized Cas9 nuclease gene driven by the 2× CaMV 35S promoter. The target sequences in both pRD213 and pRD216 can be swapped with any monocot and dicot gene target sequences. 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) and homoserine kinase (VvHSK). Plasmid pRD216 is the transient vector containing the grape HSK target sequence. The VvHSK gRNA/Cas9 cassette has been subcloned into plant expression vector pCAMBIA2300, resulting in pRD220 (the integrating vector) with kanamycin selectable marker. We have also constructed the VvHSK- and VvF3DOL-CRISPR vectors, pRD200 and pRD201 (the transient vectors), with the Arabidopsis U6 promoter driving the gRNAs. The gRNA- and Cas9-expressing cassettes in pRD200 and pRD201 were then subcloned into pCAMBIA2300, resulting in the integrating vectors pRD235 and pRD236. Since the relationship between the putative VvHSK and VvF3DOL genes and grape downy mildew (DM) resistance is largely unknown, we have used Arabidopsis thaliana (At) and its DM pathogen Hyaloperonospora parasitica as a model to study the interaction between plant and DM pathogens. We have 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. 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 showed that these At2OGFeO (pRD207) T2 mutants had much lower pathogen level than the wild type Arabidopsis Col-0 plants. For the next iteration of this project, we are in the process of carrying 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. The cDNA of these two genes have been cloned into a general plant expression vector, resulting in pRD240 and pRD241. We have transformed pRD241 (VvF3DOL cDNA) into the CRISPR-edited Arabidopsis At2OGFeO mutant plants. We have selected the homozygous lines of pRD241+/pRD207KO. We are in the process of testing the "recovery" of the susceptibility of pRD241+/pRD207KO plants to Hyaloperonospora parasitica. This data will prove that the VvF3DOL gene is involved in grape susceptibility to its downy mildew pathogen. Goal #3: Clone creeping bentgrass (Agrostis stolonifera L., As) BON1, CPK12 and DREB1C homolog genes. Using bioinformatics analysis and the existing partial EST (expressed sequence tags) library of creeping bentgrass Crenshaw cultivar, we have cloned partial cDNAs and 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. From the partially cloned gDNAs, we have identified target sequences with convenient restriction enzymatic sites. We have constructed pRD304 for AsBON1, pRD302 for AsCPK12 and pRD303 for AsDREB1C gene-editing based on pRD213. 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. pRD220 containing the VvHSK gRNA/Cas9 cassette with the ribozyme design and kanamycin selectable marker has been bombarded into NJ76 calli. However, these pRD220-transformed NJ76 calli did not survive the antibiotic selection. We have transformed NJ76 embryogenic calli with pRD216 targeting VvHSK by gene gun bombardment. The VvHSK gRNA and Cas9 will be transiently expressed without integrating into the plant genome, resulting in mutation in the VvHSK gene. It has been shown that by this gRNA/Cas9 transient expressing method, most gene-edited wheat plants did not contain the gRNA/Cas9 transgene cassettes. We have regenerated totally 11 pRD216-grape plantlets. pRD216 is a transient vector. After gene gun bombardment, the gDNA and Cas9 will be expressed and the mutation will occur in 24-48 hr (data not shown here). After plants are regenerated, this vector is not likely integrated into the grape genome. After we confirm this, we may have very likely produced first transgene-free, CRISPR-gene edited grape plants. The transient vector method will be used in the future to produce more gene-edited grape plants with DM resistance without any transgene integration to alleviate the GMO (genetically modified organism) concerns. We have also developed tissue culture protocol for creeping bentgrass (cv. Crenshaw) from mature seed-derived calli to regenerated plants. We have transformed embryogenic calli by gene gun bombardment and Agrobacterium-mediated transformation with pRD304 for AsBON1, pRD302 for AsCPK12 and pRD303 for AsDREB1C gene-editing. Transformed calli have been selected on hygromycin-containing media. 20-30 Plantlets have been regenerated from each of the three constructs' transformation events. Goal #6: Screen for gene-targeted grape and creeping bentgrass mutant plants. For the 11 pRD216-regenerated grape plants, we have isolated genomic DNAs (gDNAs) from these plants. The 473-bp DNA fragments spanning the VvHSK target site (with an XhoI restriction enzymatic site) from these 11 plants have been PCR-amplified. Restriction fragment length polymorphism (RFLP) analysis and DNA sequencing have confirmed that 4 plants have mutations at the target site of VvHSK. We are in the process of growing up these plants to be tested for downy mildew resistance. gDNA fragments spanning the target sites of AsBON1, AsCPK12 and AsDREB1C have been PCR-amplified. RFLP and sequencing analyses have been conducted. Our results showed that none of the pRD304 and pRD302 regenerants was mutant. However, totally 22 pRD303-regenerants have been shown to have various deletions at the AsDREB1C target sequence. Goal #7 testgene-edited grape and creeping bentgrassplants for disease resistance and stress tolerance. Once the VvHSK-edited grape plants grow up, we will test for their resistance to the downy mildew pathogen. A 5-day drought period was imposed on the AsDREB1C-edited plants in a growth chamber setting. Our data showed that some of the AsDREB1C mutant plants were less stressed by the drought compared to the non-gene edited plants. The AsDREB1C-edited plants and wild type (WT) Crenshaw creeping bentgrass plants were also subjected to a 50 day-salinity test. The results showed that some of the AsDREB1C-edited plants stayed greener throughout a month of salt spray compared to the WT plants.
Publications
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Grape growers, turfgrass seed producers, and turfgrass management personnel. 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, 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 continue to characterize the DM resistance of these gene-edited Arabidopsis mutants at the T2 generation with homozygous plants. We will 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: Accomplished. Goal #5: We will continue to transform NJ76 grape calli with pRD216, pRD200 and pRD201 (VvHSK- and VvF3DOL-targeting cassettes separately in a transient expressing vector) and regenerate gene-edited, transgene-free grape plants. We will transform Crenshaw calli again with the AsBON1-, AsCPK12-gene targeting constructs. Potential AsBON1, AsCPK12 mutant creeping bentgrass plants will be regenerated. Goal #6: We will characterize the future Crenshaw creeping bentgrass AsBON1-, AsCPK12-mutant plants by our established methods. Goal #7: When gene-targeted grape grow up, we will testfor their DM resistance. We will test the AsDREB1C-mutant plants for their salinity tolerance. When appropriate, we will test these gene-edited plants in the field conditions.
Impacts
What was accomplished under these goals?
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 (gRNA), and the Cas9 nuclease with monocot-specific codons driven by the maize ubiquitin promoter. For grape gene editing, 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 (the RGR design), and the humanized Cas9 nuclease gene driven by the 2× CaMV 35S promoter. The target sequences in both pRD213 and pRD216 can be swapped with any monocot and dicot gene target sequences. 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). Plasmid pRD216 is the intermediate vector (or transient vector) containing the grape HSK target sequence. The VvHSK gRNA/Cas9 cassette has been subcloned into plant expression vector pCAMBIA2300, resulting in pRD220 (the integrating vector) with kanamycin selectable marker. As a backup plan, we have also constructed the VvHSK- and VvF3DOL-CRISPR vectors, pRD200 and pRD201 (the transient vectors), with the Arabidopsis U6 promoter driving the gRNAs. The gRNA- and Cas9-expressing cassettes in pRD200 and pRD201 were then subcloned into pCAMBIA2300, resulting in the integrating vectors pRD235 and pRD236 with kanamysine selectable marker. Since the relationship between the putative VvHSK and VvF3DOL genes and grape downy mildew (DM) resistance is largely unknown, we have used Arabidopsis thaliana (At) and its DM pathogen Hyaloperonospora parasitica as a model to study the interaction between plant and DM pathogens. We have 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. 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 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. The cDNA of these two genes have been cloned into a general plant expression vector, resulting in pRD240 and pRD241. We have transformed pRD241 (VvF3DOL cDNA) into the CRISPR-edited Arabidopsis At2OGFeO mutant plants. We have selected the homozygous lines of pRD241+/pRD207KO. We are in the process of testing the "recovery" of the susceptibility of pRD241+/pRD207KO plants to Hyaloperonospora parasitica. This data will prove that the VvF3DOL gene is involved in grape susceptibility to its downy mildew pathogen. Goal #3: Clone creeping bentgrass (Agrostis stolonifera L., As) BON1, CPK12 and DREB1C homolog genes. Using bioinformatics analysis and the existing partial EST (expressed sequence tags) library of creeping bentgrass Crenshaw cultivar, we have cloned partial cDNAs and 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. From the partially cloned gDNAs, we have identified target sequences with convenient restriction enzymatic sites. We have constructed pRD304 for AsBON1, pRD302 for AsCPK12 and pRD303 for AsDREB1C gene-editing based on pRD213. 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. pRD220 containing the VvHSK gRNA/Cas9 cassette with the ribozyme design and kanamycin selectable marker has been bombarded into NJ76 calli. However, these pRD220-transformed NJ76 calli did not survive the antibiotic selection. We have transformed NJ76 embryogenic calli with pRD216 targeting VvHSK by gene gun bombardment. The VvHSK gRNA and Cas9 will be transiently expressed without integrating into the plant genome, resulting in mutation in the VvHSK gene. It has been shown that by this gRNA/Cas9 transient expressing method, most gene-edited wheat plants did not contain the gRNA/Cas9 transgene cassettes. We have regenerated totally 11 pRD216-grape plantlets. pRD216 is a transient vector. After gene gun bombardment, the gDNA and Cas9 will be expressed and the mutation will occur in 24-48 hr (data not shown here). After plants are regenerated, this vector is not likely integrated into the grape genome. After we confirm this, we may have very likely produced first transgene-free, CRISPR-gene edited grape plants. The transient vector method will be used in the future to produce more gene-edited grape plants with DM resistance without any transgene integration to alleviate the GMO (genetically modified organism) concerns. We have also developed tissue culture protocol for creeping bentgrass (cv. Crenshaw) from mature seed-derived calli to regenerated plants. We have transformed embryogenic calli by gene gun bombardment and Agrobacterium-mediated transformation with pRD304 for AsBON1, pRD302 for AsCPK12 and pRD303 for AsDREB1C gene-editing. Transformed calli have been selected on hygromycin-containing media. 20-30 Plantlets have been regenerated from each of the three constructs' transformation events. Goal #6: Screen for gene-targeted grape and creeping bentgrass mutant plants. For the 11 pRD216-regenerated grape plants, we have isolated genomic DNAs (gDNAs) from these plants. The 473-bp DNA fragments spanning the VvHSK target site (with an XhoI restriction enzymatic site) from these 11 plants have been PCR-amplified. Restriction fragment length polymorphism (RFLP) analysis and DNA sequencing have confirmed that 4 plants have mutations at the target site of VvHSK. We are in the process of growing up these plants to be tested for downy mildew resistance. gDNA fragments spanning the target sites of AsBON1, AsCPK12 and AsDREB1C have been PCR-amplified. RFLP and sequencing analyses have been conducted. Our results showed that none of the pRD304 and pRD302 regenerants was mutant. However, totally 22 pRD303-regenerants have been shown to have various deletions at the AsDREB1C target sequence. Goal #7 testgene-edited grape and creeping bentgrassplants for disease resistance and stress tolerance. Once the VvHSK-edited grape plants grow up, we will test for their resistance to the downy mildew pathogen. A 5-day drought period was imposed on the AsDREB1C-edited plants in a growth chamber setting. Our data showed that some of the AsDREB1C mutant plants were less stressed by the drought compared to the non-gene edited plants.
Publications
|
Progress 10/01/16 to 09/30/17
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?Through presentations at the recent Rutgers Turfgrass Symposium 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 continue to characterize the DM resistance of these gene-edited Arabidopsis mutants at the T2 generation with homozygous plants. We will 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: Accomplished. Goal #5: We will continue to transform NJ76 grape calli with pRD216, pRD200 and pRD201 (VvHSK- and VvF3DOL-targeting cassettes separately in a transient expressing vector) and regenerate gene-edited, transgene-free grape plants. We have transformed Crenshaw creeping bentgrass with AsBON1-, AsCPK12- andAsDREB1C-gene targeting constructs. Potential AsBON1, AsCPK12 andAsDREB1C mutant creeping bentgrass plants have been regenerated. Goal #6: When grape plantlets are regenerated from the transformed somatic embryogenic calli, they will be screened by the Genetic Analyzer (Applied Biosystems Inc.) on the PCR-amplied gDNA fragments spanning the VvHSK and VvF3DOL mutation sites. We will continue to characterize the potential Crenshaw creeping bentgrass mutant plants by sequencing the PCR-amplified gDNAs. Goal #7: When gene-targeted grape and creeping bentgrass mutant plants are obtained and confirmed, we will testthe gene-edited grape and creeping bentgrassplants for disease resistance and stress tolerance.
Impacts
What was accomplished under these goals?
CRISPR/Cas-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. For grape gene editing, 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. The target sequences in both pRD213 and pRD216 can be swapped with any monocot and dicot gene target sequences. 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). Plasmid pRD216 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 used Arabidopsis thaliana (At) and its DM pathogen Hyaloperonospora parasitica as a model to study the interaction between plant and DM pathogens. We have 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. 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. 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. From the partially cloned gDNAs of these three genes, we have identified target sequences with convenient restriction enzymatic sites. We have constructed pRD304 for AsBON1, pRD302 for AsCPK12 and pRD303 for AsDREB1C gene-editing based on pRD213. 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. pRD220 containing the VvHSK gRNA/Cas9 cassette with the ribozyme design and kanamycin selectable marker has been bombarded into NJ76 calli. However, these pRD220-transformed NJ76 calli did not survive the antibiotic selection. We have since transformed NJ76 embryogenic calli with pRD216 targeting VvHSK by gene gun bombardment. The VvHSK gRNA and Cas9 will be transiently expressed without integrating into the plant genome, resulting in mutation in the VvHSK gene. It has been shown that by this gRNA/Cas9 transient expressing method, most gene-edited wheat plants did not contain the gRNA/Cas9 transgene cassettes. This method will be used in the future to produce gene-edited grape plants with DM resistance without any transgene integration to alleviate the GMO (genetically modified organism) concerns. We have also developed tissue culture protocol for creeping bentgrass (cv. Crenshaw) from mature seed-derived calli to regenerated plants. We have transformed embryogenic calli by gene gun bombardment and Agrobacterium-mediated transformation with pRD304 for AsBON1, pRD302 for AsCPK12 and pRD303 for AsDREB1C gene-editing. Transformed calli have been selected on hygromycin-containing media. 20-30 Plantlets have been regenerated from each of the three constructs' transformation events. Goal #6: Screen for gene-targeted grape and creeping bentgrass mutant plants. The regeneration of transformed grape tissues is still ongoing. For the regenerated Crenshaw creeping bentgrass plants, we have isolated gDNAs from all the plantlets and amplified the gDNA fragments spanning the targeted mutation sites by PCR. The PCR fragments were re-PCR amplified with the same reverse primer and the forward primer with a FAM-fluorescence label. The new PCR fragments from wild type (WT) and potential mutant plants were analyzed by the Genetic Analyzer (Applied Biosystems Inc.). The nucleotide deletion or insertion in the PCR fragments from potential mutant plants were easily identified when compared to the WT PCR fragment. We have identified several mutants from each of the pRD304, pRD302 and pRD303 lines. We are in the process of further characterizing the mutations of these lines. Once they are confirmed, we will carry out the experiments in Goal #7 to test for their disease resistance and stress tolerance.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Di, R. and S. Bonos. 2017. CRISPR-gene editing of creeping bentgrass to improve stress tolerance and disease resistance. 26th Annual Rutgers Turfgrass Symposium, Jan. 13, 2017. Page 35 in the Proceedings.
|
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
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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
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