Progress 05/15/19 to 07/18/22
Outputs
Target Audience: An abstract was presented at the annual meeting of the American Pathological Society in Boston, MA that described the development of a system to infect nematodes with a cloned virus. The audience reached by the presentation was professional scientists interested and knowledgable in disease causing agents of plants. While many scientific conferences were canceled in 2020 because of the Covid-19 pandemic, we made a virtual presentation at the Digital Agronomy Day at the University of Illinois entitled "Managing Soybean Cyst Nematodes Using Genetic Nematicides" that described the goals and progress of our NIFA project. The audience reached by the presentation consisted of farmers, agricultural professional and members of the public interested in recent developments in production agriculture. Scientists interested instudying plant-nematode interactions and mechanisms of gene expression in nematodes that may have read our recently published paper on "Transient expression of a luciferase mRNA in plant-parasitic and free-living nematodes by electroporation" inthe journal Molecular and Biochemical Parasitology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? Avirtual presentation was prepared that described our project and its progress to farmers and agricultural professionals at the University of Illinois in August of 2020 and by publication of our results in a proceeding and ina scientific journal. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Major activities completed/experiments conducted for Objective 1: We showed thatviruliferous extracts prepared from sugar beet cyst nematode (SBCN) infected with SBCNV1 were successfully transferred to C. elegans, root knot nematode (RKN, Meloidogyne incognita, and soybean cyst nematode (SCN, Heterodera glycines). The virus persisted in nematode cultures for multiple generations. We also confirmed that the cloned SBCNV1 genome was biologically by inoculating RKN with in vitro transcripts of a wild-type SBCNV1 full-length clone by electroporation and then using them to inoculate tomato plants. After 3 months, SBCNV1 RNA was detected by RT-PCR in RKN eggs developed on plant roots demonstrating that the clones of SBCNV1 were fully infectious in RKN. Additional experiments were performed to optimize expression of electroporated RNAs in nematodes.We developed methods for the expression of a nematode-optimized NanoLuc luciferase mRNA in the form ofin vitrotranscripts following whole-animal electroporation ofHeterodera glycines,Meloidogyne incognita, andC.elegans. Optimization of electroporation-parameters for nematodes requires consideration of multiple factors such as the developmental stage and number of nematodes under study, type and amount of the foreign molecule(s), type of cuvette, composition and amount of electroporation buffer, electrode design, electrical field, and pulse duration. Based on bioluminescent signals from the lysed nematodes in our initial trials, we identified the optimal conditions for the electroporation parameters such as voltage (500V), pulse time 9000 ms), and capacitance (100 µF) in 2-mm cuvettes and 200 µl electroporation buffer using a PG 200 Progenetor II electroporator. Using these conditions, we successfully demonstrated the delivery of NanoLuc mRNA construct and its transient expression in J2 nematodes of RKN, SCN, and mixed life stages ofC. elegans. The ability to transiently express single-stranded RNA constructs in economically important PPN provides a rapid means to evaluate nematode and/or foreign genes for their biological significance and potential role in nematode management. Major activities completed/experiments conducted for Objective 2:Efforts to express foreign genes in the soybean cyst nematode (SCN), Heterodera glycines, were partly successful. The approach taken was to build a set of plasmid vectors that used SCN regulatory sequences derived from an abundantly expressed gene. Our project goals were to obtain stably expressed foreign genes in SCN and to develop a CRISPR-Cas9 system that would allow gene editing in our target nematode. To obtain stable expression we cloned a gene from SCN that was abundantly expressed in the epidermis of the nematode, HgFAR1, and inserted it into a plasmid vector. We found that GFP could be translationally fused to the last exon of HgFAR1 and would produce a transcript with the HgFAR1 and GFP fused together when the plasmid was bombarded into nematode cells using micro projectile bombardment. We did notice a small cryptic intron in the GFP construct, but this mis-splicing was eliminated when we removed a cryptic splice site from the gene. Nematodes bombarded with the construct produced green fluorescent signals in their epidermis, suggesting the construct was working as planned. In order to get integration of the construct into the nematodes' genome, we transferred the Hgfar1:GFP to a new plasmid so the gene fusion was flanked by piggyback transposon inverted repeats. This new construct should be integrated into the SCN genome in the presence of the appropriate transposase, which we added to another HgFar1 gene, also fused to the last exon of HgFAR1. When the HgFAR1:transposon plasmid was bombarded into SCN and the resulting mRNA analyzed for the presence of HgFAR1:transposase fusion transcripts, no full sized fusion mRNAs could be identified. We theorized that that larger transposase gene may contain several cryptic introns and thus the coding region of the transposase would need to be resynthesized to remove possible cryptic intron cleavage sites. One way around this cryptic intron issue, we theorized, may be to use a synthetic mRNA for the transposase and co-bombard this RNA with the integration HgFAR1:GFP plasmid. Initial experiments that used this approach showed enhanced GFP expression in nematode epidermal cells, but we have not yet confirmed we have constructs integrated into the SCN genome. However, since this approach looked promising, we also generated a synthetic mRNA for Cas9, which should also be translated if bombarded into the nematodes. In the Cas9 system, we also explored the ability to generate guide RNAs in SCN. To this end, we cloned the HgU6 promoter and modified it to express a guide RNA that should target the HgFAR1 mRNA. We bombarded SCN with this new U6 construct and were able to use RT-QPCR to confirm abundant expression of the guide RNA in SCN, suggesting this RNA expression system is working very well. We are currently combining the construct expressing the guide RNA with the synthetic Cas9 mRNA that can be used to edit HgFar1. Overall, we feel all components of our project are working well and we now just need to extensively test these constructs to confirm our transformation/editing system is working as planned.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Thekke-Veetil, T., McCoppin, N. K., Domier, L. L., Hajimoran, M. R., Lambert, K. N., Lim, H. S., and Hartman, G. L. 2022. Transient expression of a luciferase mRNA in plant-parasitic and free-living nematodes by electroporation. Molecular and Biochemical Parasitology https://doi: 10.1016/j.molbiopara.2022.111489.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Development of a full-length clone of sugar beet cyst nematode virus 1 and its successful introduction into soybean cyst nematode
Thanuja Thekke Veetil, Department of Crop Sciences, University of Illinois, Urbana, IL, M. R. Hajimorad, University of Tennessee, Knoxville, TN, Kris Lambert, University of Illinois, Urbana, IL and Leslie Domier, USDA ARS, Urbana, IL
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Domier, L.L., Thekke-Veetil, T., Hajimorad, M.R., and Lambert, K.N. 2020. Managing Soybean Cyst Nematodes Using Genetic Nematicides. Digital Agronomy Day Video Series, University of Illinois https://youtu.be/G6rp-rcCRjc
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Progress 05/15/21 to 05/14/22
Outputs
Target Audience:Scientists interested instudying plant-nematode interactions and mechanisms of gene expression in nematodes that read our most recent publication. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Yes, during this reporting period we published one paper on "Transient expression of a luciferase mRNA in plant-parasitic and free-living nematodes by electroporation" in the journal ofMolecular and Biochemical Parasitology. What do you plan to do during the next reporting period to accomplish the goals?The project is scheduled for termination with no future reports planned.
Impacts
What was accomplished under these goals?
Major activities completed/experiments conducted for Objective 1:Our previous report showed that viruliferous extracts prepared from sugar beet cyst nematode (SBCN) infected with SBCNV1 were successfully transferred to C. elegans, root knot nematode (RKN, Meloidogyne incognita, and soybean cyst nematode (SCN, Heterodera glycines). The virus persisted in nematode cultures for multiple generations. We also confirmed that the cloned SBCNV1 genome was biologically by inoculating RKN with in vitro transcripts of a wild-type SBCNV1 full-length clone by electroporation and then using them to inoculate tomato plants. After 3 months, SBCNV1 RNA was detected by RT-PCR in RKN eggs developed on plant roots demonstrating that the clones of SBCNV1 were fully infectious in RKN. Additional experiments were performed to optimize expression of electroporated RNAs in nematodes.We developed methods for the expression of a nematode-optimized NanoLuc luciferase mRNA in the form ofin vitrotranscripts following whole-animal electroporation ofHeterodera glycines,Meloidogyne incognita, andC.elegans. Optimization of electroporation-parameters for nematodes requires consideration of multiple factors such as the developmental stage and number of nematodes under study, type and amount of the foreign molecule(s), type of cuvette, composition and amount of electroporation buffer, electrode design, electrical field, and pulse duration. Based on bioluminescent signals from the lysed nematodes in our initial trials, we identified the optimal conditions for the electroporation parameters such as voltage (500V), pulse time 9000 ms), and capacitance (100 µF) in 2-mm cuvettes and 200 µl electroporation buffer using a PG 200 Progenetor II electroporator. Using these conditions, we successfully demonstrated the delivery of NanoLuc mRNA construct and its transient expression in J2 nematodes of RKN, SCN, and mixed life stages ofC. elegans. The ability to transiently express single-stranded RNA constructs in economically important PPN provides a rapid means to evaluate nematode and/or foreign genes for their biological significance and potential role in nematode management. Major activities completed/experiments conducted for Objective 2:Efforts to express foreign genes in the soybean cyst nematode (SCN), Heterodera glycines, were partly successful. The approach taken was to build a set of plasmid vectors that used SCN regulatory sequences derived from an abundantly expressed gene. Our project goals were to obtain stably expressed foreign genes in SCN and to develop a CRISPR-Cas9 system that would allow gene editing in our target nematode. To obtain stable expression we cloned a gene from SCN that was abundantly expressed in the epidermis of the nematode, HgFAR1, and inserted it into a plasmid vector. We found that GFP could be translationally fused to the last exon of HgFAR1 and would produce a transcript with the HgFAR1 and GFP fused together when the plasmid was bombarded into nematode cells using micro projectile bombardment. We did notice a small cryptic intron in the GFP construct, but this mis-splicing was eliminated when we removed a cryptic splice site from the gene. Nematodes bombarded with the construct produced green fluorescent signals in their epidermis, suggesting the construct was working as planned. In order to get integration of the construct into the nematodes' genome, we transferred the Hgfar1:GFP to a new plasmid so the gene fusion was flanked by piggyback transposon inverted repeats. This new construct should be integrated into the SCN genome in the presence of the appropriate transposase, which we added to another HgFar1 gene, also fused to the last exon of HgFAR1. When the HgFAR1:transposon plasmid was bombarded into SCN and the resulting mRNA analyzed for the presence of HgFAR1:transposase fusion transcripts, no full sized fusion mRNAs could be identified. We theorized that that larger transposase gene may contain several cryptic introns and thus the coding region of the transposase would need to be resynthesized to remove possible cryptic intron cleavage sites. One way around this cryptic intron issue, we theorized, may be to use a synthetic mRNA for the transposase and co-bombard this RNA with the integration HgFAR1:GFP plasmid. Initial experiments that used this approach showed enhanced GFP expression in nematode epidermal cells, but we have not yet confirmed we have constructs integrated into the SCN genome. However, since this approach looked promising, we also generated a synthetic mRNA for Cas9, which should also be translated if bombarded into the nematodes. In the Cas9 system, we also explored the ability to generate guide RNAs in SCN. To this end, we cloned the HgU6 promoter and modified it to express a guide RNA that should target the HgFAR1 mRNA. We bombarded SCN with this new U6 construct and were able to use RT-QPCR to confirm abundant expression of the guide RNA in SCN, suggesting this RNA expression system is working very well. We are currently combining the construct expressing the guide RNA with the synthetic Cas9 mRNA that can be used to edit HgFar1. Overall, we feel all components of our project are working well and we now just need to extensively test these constructs to confirm our transformation/editing system is working as planned.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Thekke-Veetil, T., McCoppin, N. K., Domier, L. L., Hajimoran, M. R., Lambert, K. N., Lim, H. S., and Hartman, G. L. 2022. Transient expression of a luciferase mRNA in plant-parasitic and free-living nematodes by electroporation. Molecular and Biochemical Parasitology https://doi: 10.1016/j.molbiopara.2022.111489.
|
Progress 05/15/20 to 05/14/21
Outputs
Target Audience:While many scientific conferences were canceled in 2020 because of the Covid-19 pandemic, we made a virtual presentation at the Digital Agronomy Day at the University of Illinois entitled "Managing Soybean Cyst Nematodes Using Genetic Nematicides" that described the goals and progressof our NIFA project.The audience reached by the presentation consisted of farmers, agricultural professional and members of the public interested in recent developments in production agriculture. Changes/Problems:Covid-19-related shelter-in-place proclamations and reduced work schedules hindered our progress on fulfilling the goals of the project. Consequently, we were not able to evaluate the pathogenicity of sugar beet cyst nematode virus 1 (SBCNV1) or to test Caenorhabditis elegans for large-scale production of SBCNV1. We hope to be able to return to more normal work schedules in 2021. The cleavage of recombinant mRNAs at cryptic introns continued to be a problem in the development of DNA-based methods for transgene expression in plant parasitic nematodes and the design of nematode expression vectors. What opportunities for training and professional development has the project provided?When shelter-in-place proclamations were issued by the state of Illinois, the postdoctoral scientist working on the project was given the opportunity to work remotely on a bioinformatic study to characterize viruses associated with soybean thrips, which are the major vector for soybean vein necrosis virus. The postdoctoral scientist was paid from a different funding source to work on the remote project. That work produced the following publication: Thekke-Veetil, T.; Lagos-Kutz, D.; McCoppin, N. K.; Hartman, G. L.; Ju, H.-K.; Lim, H.-S.; Domier, L. L., Soybean thrips (Thysanoptera: Thripidae) harbor highly diverse populations of arthropod, fungal and plant viruses. Viruses 2020, 12, 1376. How have the results been disseminated to communities of interest?As mentioned previously, a virtual presentation was prepared that described our project and its progress to farmers and agricultural professionals at the University of Illinois in August of 2020. What do you plan to do during the next reporting period to accomplish the goals?If a no-cost extension is granted and Covid-19 restrictions are relaxed, we will continue efforts to develop virus and DNA-based gene delivery systems for plant parasitic nematodes. For virus-based systems, we will focus on identifying insertion locations in the SBCNV1 genome that permit stable expression of inserted sequences. For DNA-based systems, we will focus on developing vectors where transcripts produced in vivo are not inappropriately processed because of the presence of cryptic intron splice sequences.
Impacts
What was accomplished under these goals?
Major activities completed/experiments conducted for Objective 1: We previously demonstrated that we could inoculate C. elegans, root knot nematode (RKN, Meloidogyne incognita, and soybean cyst nematode (SCN), Heterodera glycines, with viruliferous extracts prepared from sugar beet cyst nematodes SBCN) infected SBCNV1 and showed that the virus persisted in nematode cultures for multiple generations. To confirm that the cloned SBCNV1 genome was fully biological active, RKN inoculated with in vitro transcripts of a wild-type SBCNV1 full-length clone by electroporation. As negative controls, nematodes were also electroporated without added RNA and with in vitro transcripts of a mutant SBCNV1 clone where the nucleotide sequence that encodes the catalytic triad at the core of the RNA-dependent RNA polymerase had been deleted. After electroporation, nematodes were transferred to tomato plants susceptible to RKN. Three months later, SBCNV1 RNA was detected by RT-PCR in RKN eggs extracted from cultures inoculated with wild-type SBCNV1 transcripts but not from either of the negative controls. These experiments illustrate that the clones of SBCNV1 is fully infectious in RKN. Additional experiments were performed to optimize expression of electroporated RNAs in nematodes. Using luciferase reporter gene, we showed that there was a near linear response in luminescence to increasing the amounts of in vitro transcripts from 1 μg to 25 μg. We also compared the translation efficiencies of two splice leader sequences, a variant splice leader from the highly expressed SCN chorismate mutase mRNA and the SL1 canonical nematode splice leader sequence. The splice leader from chorismate mutase consistently produced higher levels of luciferase activity than the SL1 leader. To develop SBCNV1-based expression vectors, a duplicated protease cleavage site and a multiple cloning site were inserted into the SBCNV1 genome between the structural and non-structural domains. A luciferase gene was inserted into the modified SBCNV1 genome. RKN were electroporated with in vitro transcripts from the plasmid. The luciferase mRNA was used as a positive control. In the first set of experiments, luciferase activity was detected from the positive control at 24 hours after electroporation, but no luciferase activity was detected in nematodes electroporated with the modified virus. The experiments are being repeated with longer incubation times. Major activities completed/experiments conducted for Objective 2: Efforts to express foreign genes in the soybean cyst nematode (SCN), Heterodera glycines, were partly successful. The approach taken was to build a set of plasmid vectors that used SCN regulatory sequences derived from an abundantly expressed gene. Our project goals were to obtain stably expressed foreign genes in SCN and to develop a CRISPR-Cas9 system that would allow gene editing in our target nematode. To obtain stable expression we cloned a gene from SCN that was abundantly expressed in the epidermis of the nematode, HgFAR1, and inserted it into a plasmid vector. We found that GFP could be translationally fused to the last exon of HgFAR1 and would produce a transcript with the HgFAR1 and GFP fused together when the plasmid was bombarded into nematode cells using micro projectile bombardment. We did notice a small cryptic intron in the GFP construct, but this mis-splicing was eliminated when we removed a cryptic splice site from the gene. Nematodes bombarded with the construct produced green fluorescent signals in their epidermis, suggesting the construct was working as planned. In order to get integration of the construct into the nematodes' genome, we transferred the Hgfar1:GFP to a new plasmid so the gene fusion was flanked by piggyback transposon inverted repeats. This new construct should be integrated into the SCN genome in the presence of the appropriate transposase, which we added to another HgFar1 gene, also fused to the last exon of HgFAR1. When the HgFAR1:transposon plasmid was bombarded into SCN and the resulting mRNA analyzed for the presence of HgFAR1:transposase fusion transcripts, no full sized fusion mRNAs could be identified. We theorized that that larger transposase gene may contain several cryptic introns and thus the coding region of the transposase would need to be resynthesized to remove possible cryptic intron cleavage sites. One way around this cryptic intron issue, we theorized, may be to use a synthetic mRNA for the transposase and co-bombard this RNA with the integration HgFAR1:GFP plasmid. Initial experiments that used this approach showed enhanced GFP expression in nematode epidermal cells, but we have not yet confirmed we have constructs integrated into the SCN genome. However, since this approach looked promising, we also generated a synthetic mRNA for Cas9, which should also be translated if bombarded into the nematodes. In the Cas9 system, we also explored the ability to generate guide RNAs in SCN. To this end, we cloned the HgU6 promoter and modified it to express a guide RNA that should target the HgFAR1 mRNA. We bombarded SCN with this new U6 construct and were able to use RT-QPCR to confirm abundant expression of the guide RNA in SCN, suggesting this RNA expression system is working very well. We are currently combining the construct expressing the guide RNA with the synthetic Cas9 mRNA and plan to have evidence of HgFar1 editing in the near future. Overall, we feel all components of our project are working well and we now just need to extensively test these constructs to confirm our transformation/editing system is working as planned.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Domier, L.L., Thekke-Veetil, T., Hajimorad, M.R., and Lambert, K.N. 2020. Managing Soybean Cyst Nematodes Using Genetic Nematicides. Digital Agronomy Day Video Series, University of Illinois https://youtu.be/G6rp-rcCRjc
|
Progress 05/15/19 to 05/14/20
Outputs
Target Audience:An abstract was presented at the annual meeting of the American Pathological Society in Boston, MA that described the development of a system to infect nematodes with a cloned virus. The audience reached by the presentation was professional scientists interested and knowledgable in disease causing agents of plants Changes/Problems:The major problem we encountered was the missplicing of synthetic mRNAs because of cryptic introns. To streamline analysis of synthetic gene constructs, we have developed a rapid protocol to assay gene function based on whole-nematode electroporation. The ability to more quickly assay gene function and analysis of misspliced transcripts should allow us to designed constructs that will be expressed at higher levels in plant parasitic nematodes. What opportunities for training and professional development has the project provided?This project has given the postdoctoral scientist hired on the grant the opportunity to develop skills in the manipulation and analysis of nematode-infecting viruses. The project is one of the few projects in the world actively involved in research on nematode viruses. How have the results been disseminated to communities of interest?As mentioned previously, an abstract describing our progress with developing a reverse genetic system for a nematode-infecting virus was rpesented at the annual meeting of the American Phytopalthological Society meeting in Boston, MA in Agust 2019 What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will use the systems we developed to introduce nucleic acids into whole nematodes to assay modified virus genomes nematode gene expression constructsfor function in nematodes. We hope to overcome the problems encountered with missplicing of exogenous RNAs by removing cryptic introns from sequences prior to gene synthesis.
Impacts
What was accomplished under these goals?
Project Objectives Objective 1: Construct virus-based systems to express and silence genes in SCN. Subobjective 1A. Determine the pathogenicity of sugar beet cyst nematode virus 1 (SBCNV1) on SCN and RKN Subobjective 1B. Test Caenorhabditis elegans for large-scale production of SBCNV1 Subobjective 1C. Construct virus vectors for expression of cloned sequences in nematodes Major activities completed/experiments conducted for Objective 1: We successfully inoculated C. elegans, RKN and SCN with viruliferous extracts prepared for sugar beet cyst nematodes SBCN) infected SBCNV1. SBCNV1 was maintained in the three species of nematodes for multiple passages. In addition, SBCNV1 was detected in aerial portions of tomato plants infested with SBCNV1-infected RKN. Once cultures are established of C. elegans, RKN and SCN that are uniformly infected with SBCNV1, the pathogenicity of the virus on the nematodes will be assayed. Full-length clones of the SBCNV1 genome were modified to express a green fluorescent protein gene. Following bombardment of SCN with gold particles coated with in vitro transcripts of the modified virus, nematodes displaying green fluorescence were observed under fluorescence microscopy. Results from the experiments were presented at the annual meeting of the American Phytopathological Society in Boston, MA. To generate a quantifiable assay for levels of gene expression from the recombinant virus, the NanoLuc luciferase gene was inserted between the structural and nonstructural protein domains of SBCNV1. That construct has not been assayed yet. Objective 2: Develop DNA-based methods for transgene expression in SCN. Subobjective 2A. Develop new expression vectors Subobjective 2B. Develop high-throughput methods to identify transformed nematodes Major activities completed/experiments conducted for Objective 2: In the first set of plasmid expression vectors, the Hg-Far1 promoter and terminator sequences were used to drive a codon optimized GFP gene with synthetic introns. However, transcripts from the construct were aberrantly processed by the removal of cryptic intron sequences. To circumvent the problem, the GFP coding sequence preceded by a picornavirus 2A 'cleavage' sequence were cloned in-frame with the 5' half of the GF-Far1 gene. The hypothesis was that the natural introns present in the Hg-Far1 gene would facilitate proper processing of the mRNA and that the 2A sequence would liberate and intact GFP protein, However, the fused mRNA was still aberrantly processed. As described below, attempts are being made to preemptively identify cryptic intron sequences and remove them before gene synthesis. To generate DNA-based methods for inoculation of nematodes with cloned viruses, the bacteriophage T7 RNA polymerase gene was codon-optimized and synthetic introns were inserted into the coding sequence to enhance expression in nematodes and cloned downstream of the Hg Far1 promoter. When nematodes were bombarded with gold particles coated with the construct, T7 RNA polymerase transcripts were detected, but cryptic introns had been spliced out of the transcript disabling the gene. The T7 RNA polymerase gene was redesigned to remove candidate cryptic introns and resynthesized. The gene has not been assayed yet. Because of difficulties with cryptic introns in multiple constructs, we evaluated whole-nematode electroporation to more quickly and quantitatively assay the functionality of constructs. For those experiments, we constructed a NanoLuc luciferase gene with the 5' and 3' noncoding sequences from the highly expressed SCN chorismite mutase gene and cloned the sequence downstream of a T7 RNA polymerase promoter. Using different electroporation conditions, the optimum was selected (500 volts, 100 µF) for electroporation of in vitro transcripts of the luciferase gene produced signals about 50-fold above background after 24 hr in C.elegans, RKN and SCN. Significant luciferase activity was still detected at 72 hr after electroporation, but the highest readings were after 24 hr. We plan to use the selected electroporation conditions to assay promoter-gene for functionality in whole nematodes.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Development of a full-length clone of sugar beet cyst nematode virus 1 and its successful introduction into soybean cyst nematode
Thanuja Thekke Veetil, Department of Crop Sciences, University of Illinois, Urbana, IL, M. R. Hajimorad, University of Tennessee, Knoxville, TN, Kris Lambert, University of Illinois, Urbana, IL and Leslie Domier, USDA ARS, Urbana, IL
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