Progress 09/01/19 to 10/31/20

Outputs
Target Audience:Cover crop farmers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has allowed postdoctoral fellows Dr. Siva Velivelli and Dr. Hui Li to familiarize themselves with the fungal pathogens of pennycress A. japonica and R. solani. They have learned how to grow these pathogens and use them to infect pennycress plants. In addition, they have learned how to purify antifungal peptides, test them for in vitro antifungal activity against these peptides and generate chimeric gene constructs for expression in transgenic pennycress. By participation in weekly meetings to discuss progress against goals, Dr. Velivelli and Dr. Li have also been able to enhance their oral communication and interpersonal skills. Raymond Kennedy has been able to participate in the implementation of fungal pathogen assays intended to screen a diverse library of germplasm for disease resistance. He has furthered his work facilitating the transformation of pennycress in a highthroughput pipeline, including the rapid detection of transformants, in order to generate transgenic plants used to assess the effect of the expression of plant defensins. Max McDonald has been able to continually advance his capabilities in Agrobacterium tumafaciens preparation, as well as his skill in efficient floral dip infiltration of pennycress. How have the results been disseminated to communities of interest?The preliminary results from this project have been communicated to the CEO of CoverCress. What do you plan to do during the next reporting period to accomplish the goals?Resistance to biotic stress is a critical agronomic trait for successful introduction of pennycress on a commercial scale. Results achieved during the 5.5 months of Phase I research have laid the foundation for making significant progress toward engineering this trait in pennycress. During the proposed period of Phase II research, we will specifically focus on characterizing transgenic pennycress lines expressing antifungal peptides with direct antifungal activity and pennycress elicitor peptides capable of activating a suite of defense responses in pennycress. Further, optimized expression of genes encoding these two classes of peptides with different modes of action will lead to robust, sustainable resistance to fungal pathogens. We will utilize in vitro and in planta assays to select pennycress lines that will be used to enter in the commercialization phase of this project. Specific Aims of the proposal are: Aim1 Characterize T1 transgenic pennycress lines constitutively expressing OeDef1, NCR044_v2, PDF2.2 and PDF4.3 to identify homozygous lines and determine expression of each peptide. Expression and gene copy analyses of OeDef1, NCR044_v2_PDF2.2 and PDF4.3 in transgenic pennycress lines. Disease resistance evaluation of transgenic lines. Aim2. Characterize T1 transgenic pennycress lines expressing OeDef1, NCR044_v2, PDF2.2 and PDF4.3 from the epidermis-specific promoter or pathogen-responsive PDF1.2 promoter. Expression and gene copy analyses of OeDef1, NCR044_v2_PDF2.2 and PDF4.3 in transgenic pennycress lines. Disease resistance evaluation of transgenic lines. Aim3. Generate transgenic pennycress lines expressing TaPep1, TaPep2 and TaPep3 constitutively and test the lines for resistance to fungal pathogens. Expression of TaPep1, TaPep2 and TaPep3 in transgenic pennycress Determine JA-responsive defense gene expression in transgenic Pep lines Determine resistance of homozygous Pep lines to fungal pathogens Aim4. Identify pathogen-responsive genes in pennycress and characterize their promoters. The successful completion of these objectives will demonstrate that expression of peptides with potent antifungal activity and Peps that act as defense signaling molecules will confer broad-spectrum resistance to fungal pathogens in pennycress. Further, pathogen-responsive expression of these peptides in transgenic pennycress will confer disease resistance without compromising yield and winter hardiness.

Impacts
What was accomplished under these goals? Generate transgenic pennycress lines expressing plant defensins MtDef5 fromMedicago truncatulaand OeDef1.1 fromOlea europaeafor resistance to Alternaria black spot andRhizoctoniacrown rot. Aim1.Determinein vitroantifungal activity of antifungal plant defensins OeDef1, NCR044_v2 and novel cationic pennycress defensins PDF2.2 and PDF4.3 againstAlternaria japonicaandRhizoctonia solani. 1a. Recombinant expression and purification of antifungal peptides OeDef1, NCR044_v2, PDF2.2 and PDF4.3 The amino acid sequences of OeDef1, NCR044_v2 and the pennycress defensins, PDF2.2 and PDF4.3, are shown in Table 1. Genes encoding these peptides were custom-synthesized by Genscript, Inc (Piscataway, NJ) and cloned into pPICZα-A integration vector for expression inP. pastoris[12]. Each peptide was purified from the culture medium using the CM-Sephadex C-25 cation exchange Fast ProteinLiquid Chromatography (AKTA FPLC, GE Healthcare, USA) and C-18 Reverse Phase-High Performance Liquid Chromatography (RP-HPLC,Beckman Coulter, USA) and subsequently lyophilized as previously described [12, 31]. The purity and mass of each peptide were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and direct infusion-mass spectrometry, respectively. Table 1. Amino acid sequences and net charge of antifungal peptides studied in Phase I. Peptide Net charge Amino acid sequence OeDef1 +8 KPCTKLSKGWRGLCAPHKCSSYCIHHEGAYHGACLKNRHSKHYGCYCYYRHCY NCR044_v2 +9 AFCIQLSKPCISDKECSIVKNYRARCRKGYCVRRRIRC TaPDF2.2 +7 RTCESKSHRFKGLCLSETNCKNVCHNEGFGGGNCRGARRRCFCTRHC TaPDF4.3 +7 QMCEAKSLNFRGMCMKWRTCKFVCLSEGFTDGRCKGFIRHCICRKPCMVST Table 2. In vitroantifungal activity of PDF2.2, PDF4.3, OeDef1, and NCR044_v2 againstAlternaria japonicaandRhizoctonia solani.ND=Not Determined. Peptide Alternaria japonica -IC50(µM)/MIC (µM) Rhizoctonia solani -MIC (µM) PDF2.2 2.8 / 5.5 6 PDF4.3 2.5 / 5.1 >6 OeDef1 ND / ND 6 NCR044_v2 2.6 / 5.2 ND 1b.Determinein vitroantifungal activity of OeDef1, NCR044_v2, PDF2.2 and PDF4.3 againstAlternaria japonicaandRhizoctonia solani. The antifungal activity assay of PDF2.2, PDF4.3, OeDef1 and NCR044_v2 was conducted in 96-well microtiter plate using the mycelial fragments ofA. japonicaandR. solani. Briefly, fifty microliters of each protein dilution (0, 0.3, 0.7, 1.5, 3, 6 and 12 µM) was added to each well of the microtiter plate containing 50 µL of 5 × 104/ml mycelial suspension. The plates were incubated at room temperature, and the quantitative fungal growth inhibition was determined by measuring the absorbance at 595 nm using a (Tecan Infinite M200 ProTecan Systems Inc., San Jose, CA)microplate reader at 48 h. The fungal cell viability/cell death was determined by the resazurin cell viability assay. After incubation of the pathogen/peptide mixture for 48 h, 10 µl of resazurin solution at 0.1% (w/v) was added to each well and re-incubated overnight at room temperature. The color change from blue to pink/colorless indicated the reduction of resazurin by the presence of live fungal cells. The IC50(concentration of peptide required for 50% growth inhibition) and minimal inhibitory concentration (MIC, concentration of peptide required for complete inhibition of fungal growth) values for each peptide/A. japonicaand MIC values for each peptide/R. solanicombination are shown in Table 2. The results show that all peptides inhibit fungal growth at low micromolar concentrations. Based on these results, we have prioritized all four peptides for constitutive, tissue-specific and pathogen-responsive expression in transgenic pennycress and testing the lines for resistance. Since, transformation of pennycress is highly efficient at CoverCress, we decided to test antifungal activity of all four antifungal peptides directly in transgenic pennycress. Aim2. GenerateAgrobacterium tumefaciensexpression vectors for expression of OeDef1, NCR044_v2, PDF2.2 and PDF4.3 using constitutive, epidermis-specific and pathogen-inducible promoters in transgenic pennycress. 2a.Constitutive gene expression in pennycress.We have generated chimeric genes in which expression of OeDef1, NCR044_v2, PDF2.2 and PDF4.3 has been placed under the control of the constitutive eCaMV35S promoter. These chimeric genes have been introduced into pennycress using the floral dip transformation method. Each chimeric gene construct also contains DsRed fluorescent protein as a reporter gene to enable easy visualization and sorting of transgenic seed from null segregant seed. We have obtained DsRed+ T3 lines expressing containing the OeDef1gene construct. We have tested these lines for expression of the OeDef1 protein by Western blot analysis. We have determined that lines B28663C-6, BC28663-9 and B28663C-10 are expressing the OeDef1 protein. We have challenged line BC28663-9 with a fungal pathogen Botrytis cinerea which causes gray mold disease in several plant species including pennycress.This line exhibits significantly reduced gray mold disease symptoms than the DsRed- null line. 2b.Pathogen-responsive gene expression in pennycress.PDF1.2promoter is a pathogen-responsive promoter fromA. thaliana. It is known to be induced byA. brassicicola[40]. We have generated chimeric gene constructs in which expression of PDF2.2, PDF4.3, OeDef1 and NCR044_v2 has been placed under the control of this promoter. SinceA. thalianaand pennycress are closely related, we expect thePDF1.2promoter to be pathogen-responsive in pennycress. The current status of transformation events obtained using the PDF1.2 promoter/gene constructs is summarized in Table 3. 2c. Epidermis-specific gene expression in pennycress ArabidopsisA14promoter is expressed specifically in epidermis [38, 39]. We expect that epidermis-specific expression of PDF2.2, PDF4.3, OeDef1 and NCR044_v2 will prevent fungal spore germination at the first site of contact in epidermis. Therefore, we have generated chimeric gene construct for expression of each peptide under the control ofA14promoter. The current status of transformation events obtained using theA14promoter/gene constructs is summarized in Table 3. Aim3. Identification of pathogen-responsive promoters of pennycress Our aim is to express potent antifungal peptides from a pathogen-inducible promoter in transgenic pennycress to minimize any potential impact on the agronomic traits of pennycress in the field. We have screened a number of pennycress genotypes for susceptibility to the field isolate ofA. japonica. Six detached leaves of these genotypes were inoculated with 5X 105mycelial fragments per ml of this pathogen and disease lesions were observed 3 days post-inoculation (not shown). Y1126 genotype was identified as most susceptible and B-14 as most resistant genotype in this assay. In Phase II, we will confirm this phenotype in whole plants challenged with this pathogen in a growth chamber. If this phenotype is confirmed at the whole plant level, transcriptome study will be conducted to identify genes that are activated rapidly during infection by this pathogen in Y1126. The promoters of genes rapidly activated by this pathogen will be excellent candidates to drive the expression of antifungal peptides in transgenic pennycress.

Publications

Progress 09/01/19 to 08/31/20

Outputs
Target Audience:Cover crop farmers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has allowed postdoctoral student Dr. Siva Velivelli to familiarize himself with the fungal pathogens of pennycress A. japonica and R. solani. He has learned how to grow these pathogens and use them to infect pennycress plants. In addition, he has learned how to purify antifungal peptides and test them for in vitro antifungal activity against these peptides. By participation in weekly meetings to discuss progress against goals, Dr. Velivelli has also been able to enhance his oral communication and interpersonal skills. Raymond Kennedyhas been able to participate inthe implementation of fungal pathogen assays intended to screen a diverse library of germplasm for disease resistance. He has furthered his workfacilitating the transformation of pennycress in a high-throughput pipeline, including the rapid detection of transformants,in order to generate transgenic plants used to assess the effect of the expression of plant defensins. Max McDonald has been able to continually advance his capabilities in Agrobacterium tumafaciens preparation, as well as his skill in efficient floral dip infiltration of pennycress. How have the results been disseminated to communities of interest?The preliminary results from this project have been communicated to the CEO of CoverCress. What do you plan to do during the next reporting period to accomplish the goals? We will implement quantitative disease resistance assays to evaluate antifungal activity of various peptides against A. japaonica and Rhizoctonia solani using pennycress plants in a growth chamber. Transgenic lines containing the chimeric gene constructs will be characterized for copy number and expression of the gene as well as zygosity. Homozygous transgenic lines for each chimeric gene construct will be evaluated for resistance to A. japonica and R. solani in a growth chamber.

Impacts
What was accomplished under these goals? 1. Generate transgenic pennycress lines expressing plant defensins MtDef5 from Medicago truncatula and OeDef1.1 from Olea europaea for resistance to Alternaria black spot and Rhizoctonia crown rot. 1a. We generated Agrobacterium tumefaciens vectors in which MtDef5, OeDef1 and OeDef1_v1 genes were each placed under the control of the E35S promoter. These vectors also contain the DsRed reporter gene. Vector containing each chimeric gene was introduced into pennycress using the floral dip transformation method. MtDef5 construct was introduced into 3 genotypes of pennycress. OeDef1 and OeDef1_v1 constructs were each introduced into two genotypes of pennycress. We have identified DsRed positive T1 seed for each construct. The DsRed+ seed will be planted and the T2 progeny will be screened for identification of the homozygous lines. 1b. Homozygous lines will be tested for expression of MtDef5, OeDef1 and OeDef1_v1 by Western blot analysis. We have obtained polyclonal antibody for MtDef5 and OeDef1 to perform this analysis. 1c. Homozygous lines expressing MtDef5, OeDef1 or OeDef1_v1 will be tested for resistance to Alternaria japonica and Rhizoctonia solani in coming months. 2. Determine in vitro antifungal activity of cationic pennycress defensins against the target pathogens. 2a. Express genes encoding pennycress defensins PDF2.2, PDF4.3 and PDF4.6 in Pichia pastoris and purify each defensin to homogeneity. Genes encoding PDF2.2, PDF4.3 and PDF4.6 were custom-synthesized by Genscript, Inc (Piscataway, NJ) and cloned into pPICZα-A integration vector for expression in P. pastoris. Each peptide was purified from the culture medium using the CM-Sephadex C-25 cation exchange Fast ProteinLiquid Chromatography (AKTA FPLC, GE Healthcare, USA) and C-18 Reverse Phase-High Performance Liquid Chromatography (RP-HPLC, Beckman Coulter, USA) and subsequently lyophilized as previously described. The peptide was dissolved in nuclease-free water. The peptide concentration was determined by using a Nanodrop 2000c (Thermo Scientific, USA) at A280 based on the molar extinction coefficient (3230 M-1cm-1) and molecular weight of each peptide. The purity and mass of each peptide was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and direct infusion-mass spectrometry, respectively. 2b. Determine in vitro antifungal activity of PDF2.2, PDF4.3 and PDF4.6 against Alternaria japonica and Rhizoctonia solani. The antifungal activity assay of PDF2.2, PDF4.3 and PDF4.6 was conducted in 96-well microtiter plate using the mycelial fragments of A. japonica and R. solani. Briefly, fifty microliters of each protein dilution (0, 0.3, 0.7, 1.5, 3, 6 and 12 µM) was added to each well of the microtiter plate containing 50 µL of 5 × 104/ml mycelial suspension. The plates were incubated at room temperature, and the quantitative fungal growth inhibition was determined by measuring the absorbance at 595 nm using a (Tecan Infinite M200 ProTecan Systems Inc., San Jose, CA) microplate reader at 48 h. The fungal cell viability/cell death was determined by the resazurin cell viability assay. After incubation of the pathogen/peptide mixture for 48 h, 10 µl of resazurin solution at 0.1% (w/v) was added to each well and re-incubated overnight at room temperature. The color change from blue to pink/colorless indicated the reduction of resazurin by the presence of live fungal cells. The IC50 (concentration of peptide required for 50% growth inhibition) and minimal inhibitory concentration (MIC, concentration of peptide required for complete inhibition of fungal growth) values for each peptide/A. japonica and MIC values for each peptide/R. solani combination are shown in Table 1. Table 1.In vitroantifungal activity of PDF2.2, PDF2.2_v1, PDF4.3 and PDF4.6 againstAlternaria japonicaandRhizoctonia solani Peptide Alternaria japonica IC50 (µM) Alternaria japonica MIC (µM) Rhizoctonia solani MIC (µM) PDF2.2 2.79 5.50 6 PDF2.2v1 4.40 5.74 >6 PDF4.3 2.54 5.12 >6 PDF4.6 4.35 5.70 >6 2c. Generate Agrobacterium tumefaciens expression vectors for constitutive expression of PDF2.2, PDF4.3 and PDF4.6 and initiate pennycress transformation. Based on the in vitro antifungal activity of the three peptides tested, we have prioritized genes encoding PDF2.2 and PDF4.3 for transformation into pennycress. We have generated chimeric genes in which expression of PDF2.2 and PDF4.3 has been placed under the control of the constitutive e35S promoter. These chimeric genes have been introduced into two different genotypes of pennycress. The T2 seed segregating for each gene will be available for further characterization in March/April. 3. Identify pathogen-responsive genes in pennycress and characterize their promoters. 3a. Arabidopsis PDF1.2 promoter is known to be induced by Alternaria brassicicola. It is expected to be pathogen-inducible in pennycress. Arabidopsis PDF1.2 promoter is known to be induced by fungal pathogens. We have generated chimeric gene constructs in which expression of PDF2.2, OeDef1_v1, NCR2_v2 and PDF4.3 has been placed under the control of this promoter. Each chimeric gene has been introduced into two independent genotypes of pennycress using the floral dip transformation method. T1 seed for each chimeric gene construct will be available in April/May. 3b. Use Arabidopsis epidermis-specific A14 promoter for expression of PDF2.2, OeDef1_v1, NCR2_v2 and PDF4.3 in pennycress. Arabidopsis A14 promoter is expressed specifically in epidermis. We expect that epidermis-specific expression of PDF2.2, OeDef1_v1, NCR2_v2 and PDF4.3 will prevent fungal spore germination at the first site of contact in epidermis. Therefore, we have generated chimeric gene construct for expression of each peptide under the control of A14 promoter. These chimeric gene constructs have been introduced into two genotypes of pennycress using floral dip method. T0 seed for each chimeric gene construct will be available in April/May.

Publications

Progress 04/01/19 to 03/31/20

Outputs
Target Audience:Cover crop farmers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has allowed postdoctoral student Dr. Siva Velivelli to familiarize himself with the fungal pathogens of pennycress A. japonica and R. solani. He has learned how to grow these pathogens and use them to infect pennycress plants. In addition, he has learned how to purify antifungal peptides and test them for in vitro antifungal activity against these peptides. By participation in weekly meetings to discuss progress against goals, Dr. Velivelli has also been able to enhance his oral communication and interpersonal skills. Raymond Kennedyhas been able to participate inthe implementation of fungal pathogen assays intended to screen a diverse library of germplasm for disease resistance. He has furthered his workfacilitating the transformation of pennycress in a high-throughput pipeline, including the rapid detection of transformants,in order to generate transgenic plants used to assess the effect of the expression of plant defensins. Max McDonald has been able to continually advance his capabilities in Agrobacterium tumafaciens preparation, as well as his skill in efficient floral dip infiltration of pennycress. How have the results been disseminated to communities of interest?The preliminary results from this project have been communicated to the CEO of CoverCress. What do you plan to do during the next reporting period to accomplish the goals? We will implement quantitative disease resistance assays to evaluate antifungal activity of various peptides against A. japaonica and Rhizoctonia solani using pennycress plants in a growth chamber. Transgenic lines containing the chimeric gene constructs will be characterized for copy number and expression of the gene as well as zygosity. Homozygous transgenic lines for each chimeric gene construct will be evaluated for resistance to A. japonica and R. solani in a growth chamber.

Impacts
What was accomplished under these goals? 1. Generate transgenic pennycress lines expressing plant defensins MtDef5 from Medicago truncatula and OeDef1.1 from Olea europaea for resistance to Alternaria black spot and Rhizoctonia crown rot. 1a. We generated Agrobacterium tumefaciens vectors in which MtDef5, OeDef1 and OeDef1_v1 genes were each placed under the control of the E35S promoter. These vectors also contain the DsRed reporter gene. Vector containing each chimeric gene was introduced into pennycress using the floral dip transformation method. MtDef5 construct was introduced into 3 genotypes of pennycress. OeDef1 and OeDef1_v1 constructs were each introduced into two genotypes of pennycress. We have identified DsRed positive T1 seed for each construct. The DsRed+ seed will be planted and the T2 progeny will be screened for identification of the homozygous lines. 1b. Homozygous lines will be tested for expression of MtDef5, OeDef1 and OeDef1_v1 by Western blot analysis. We have obtained polyclonal antibody for MtDef5 and OeDef1 to perform this analysis. 1c. Homozygous lines expressing MtDef5, OeDef1 or OeDef1_v1 will be tested for resistance to Alternaria japonica and Rhizoctonia solani in coming months. 2. Determine in vitro antifungal activity of cationic pennycress defensins against the target pathogens. 2a. Express genes encoding pennycress defensins PDF2.2, PDF4.3 and PDF4.6 in Pichia pastoris and purify each defensin to homogeneity. Genes encoding PDF2.2, PDF4.3 and PDF4.6 were custom-synthesized by Genscript, Inc (Piscataway, NJ) and cloned into pPICZα-A integration vector for expression in P. pastoris. Each peptide was purified from the culture medium using the CM-Sephadex C-25 cation exchange Fast ProteinLiquid Chromatography (AKTA FPLC, GE Healthcare, USA) and C-18 Reverse Phase-High Performance Liquid Chromatography (RP-HPLC, Beckman Coulter, USA) and subsequently lyophilized as previously described. The peptide was dissolved in nuclease-free water. The peptide concentration was determined by using a Nanodrop 2000c (Thermo Scientific, USA) at A280 based on the molar extinction coefficient (3230 M-1cm-1) and molecular weight of each peptide. The purity and mass of each peptide was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and direct infusion-mass spectrometry, respectively. 2b. Determine in vitro antifungal activity of PDF2.2, PDF4.3 and PDF4.6 against Alternaria japonica and Rhizoctonia solani. The antifungal activity assay of PDF2.2, PDF4.3 and PDF4.6 was conducted in 96-well microtiter plate using the mycelial fragments of A. japonica and R. solani. Briefly, fifty microliters of each protein dilution (0, 0.3, 0.7, 1.5, 3, 6 and 12 µM) was added to each well of the microtiter plate containing 50 µL of 5 × 104/ml mycelial suspension. The plates were incubated at room temperature, and the quantitative fungal growth inhibition was determined by measuring the absorbance at 595 nm using a (Tecan Infinite M200 ProTecan Systems Inc., San Jose, CA) microplate reader at 48 h. The fungal cell viability/cell death was determined by the resazurin cell viability assay. After incubation of the pathogen/peptide mixture for 48 h, 10 µl of resazurin solution at 0.1% (w/v) was added to each well and re-incubated overnight at room temperature. The color change from blue to pink/colorless indicated the reduction of resazurin by the presence of live fungal cells. The IC50 (concentration of peptide required for 50% growth inhibition) and minimal inhibitory concentration (MIC, concentration of peptide required for complete inhibition of fungal growth) values for each peptide/A. japonica and MIC values for each peptide/R. solani combination are shown in Table 1. Table 1.In vitroantifungal activity of PDF2.2, PDF2.2_v1, PDF4.3 and PDF4.6 againstAlternaria japonicaandRhizoctonia solani Peptide Alternaria japonica IC50 (µM) Alternaria japonica MIC (µM) Rhizoctonia solani MIC (µM) PDF2.2 2.79 5.50 6 PDF2.2v1 4.40 5.74 >6 PDF4.3 2.54 5.12 >6 PDF4.6 4.35 5.70 >6 2c. Generate Agrobacterium tumefaciens expression vectors for constitutive expression of PDF2.2, PDF4.3 and PDF4.6 and initiate pennycress transformation. Based on the in vitro antifungal activity of the three peptides tested, we have prioritized genes encoding PDF2.2 and PDF4.3 for transformation into pennycress. We have generated chimeric genes in which expression of PDF2.2 and PDF4.3 has been placed under the control of the constitutive e35S promoter. These chimeric genes have been introduced into two different genotypes of pennycress. The T2 seed segregating for each gene will be available for further characterization in March/April. 3. Identify pathogen-responsive genes in pennycress and characterize their promoters. 3a. Arabidopsis PDF1.2 promoter is known to be induced by Alternaria brassicicola. It is expected to be pathogen-inducible in pennycress. Arabidopsis PDF1.2 promoter is known to be induced by fungal pathogens. We have generated chimeric gene constructs in which expression of PDF2.2, OeDef1_v1, NCR2_v2 and PDF4.3 has been placed under the control of this promoter. Each chimeric gene has been introduced into two independent genotypes of pennycress using the floral dip transformation method. T1 seed for each chimeric gene construct will be available in April/May. 3b. Use Arabidopsis epidermis-specific A14 promoter for expression of PDF2.2, OeDef1_v1, NCR2_v2 and PDF4.3 in pennycress. Arabidopsis A14 promoter is expressed specifically in epidermis. We expect that epidermis-specific expression of PDF2.2, OeDef1_v1, NCR2_v2 and PDF4.3 will prevent fungal spore germination at the first site of contact in epidermis. Therefore, we have generated chimeric gene construct for expression of each peptide under the control of A14 promoter. These chimeric gene constructs have been introduced into two genotypes of pennycress using floral dip method. T0 seed for each chimeric gene construct will be available in April/May.

Publications