Progress 07/01/20 to 06/30/24
Outputs
Target Audience:Researchers interested in using viruses for gene editing or other gene function analysis applications, ag biotechnologyindustry, andcommodity groups. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A postdoctoral scientist and two different graduate students have participated in this project. They had the experience of working in a collaborative project with scientists from different labs, gained experience in gene editing and virus-based delivery technologies, and participants were exposed to working with different crop plants including sorghum, switchgrass, and barley. The postdoctoral scientist attended a meeting where she could present results from the work and network with scientists interested in applications for the foxtail mosaic virus in sorghum. How have the results been disseminated to communities of interest?We published three papers that were listed in the 2022 and 2023 annual reports. Two were research articles in which we investigated the potential roles of different RNA mobility signals in promoting gene editing in the context of foxtail mosaic virus, and in the other, we established foxtail mosaic virus is a useful tool for virus-induced gene silencing for testing gene function in sorghum. The third paper was a comprehensive review on foxtail mosaic virus and its applications in plant research. We presented a poster at the 2023 Plant and Animal Genome meeting in San Diego, CA in which results from foxtail mosaic virus VIGS studies in sorghum were presented. We expect to submit at least one more publication based on this support in which we used foxtail mosaic virus induced gene silencing to study the function of a gene involved in sorghum photosynthesis. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In this project, we aimed to investigate the use of foxtail mosaic virus for gene editing in sorghum and switchgrass. In the course of initiating work in sorghum, we established that foxtail mosaic virus is a useful tool for doing virus-induced gene silencing studies in sorghum. While brome mosaic virus had previously been shown by others to induce gene silencing in sorghum, it has not been adopted by the field for this use, and the growth conditions are not optimal for sorghum. We found that foxtail mosaic virus induces gene silencing under conditions that are more physiologically relevant to sorghum and that silencing is compatible with different kinds of assays, including pathogen defense (published) and physiological assays (e.g., photosynthesis measurements). We are working on a manuscript that includes the application of foxtail mosaic virus for studying functions of genes affecting photosynthesis and intend to submit it in 2025. We investigated the functionality of different RNA mobility signals, including the mobility sequence from Arabidopsis thaliana Flower Locus T (AtFT), FT homologs from maize, and tRNA sequences in foxtail mosaic virus. The AtFT and AttRNA sequences were shown by others to promote heritable gene editing when expressed from tobacco rattle virus and potato virus X in Nicotiana benthamiana. We found that such sequences could slightly promote gene editing in the leaves of Nicotiana benthamiana and maize in the context of foxtail mosaic virus, but they could not cause foxtail mosaic virus to gain the ability to induce heritable genome edits. Our current hypothesis is that the mobility signals can enhance the ability of viruses to induce heritable gene edits in certain combinations of virus and host in which the virus may already possess the ability to access the shoot apical meristem. We tested somatic gene editing in sorghum and switchgrass using foxtail mosaic virus to deliver single guide RNAs targeting phytoene desaturase genes (pds). The sorghum and switchgrass lines had been previously transformed to express the Cas9 protein. In the sorghum Cas9 plants, we found that the guide RNAs in foxtail mosaic virus were deleted very quickly, and thus we were not successful in identifying somatic edits. We explored the use of alternative viruses including brome mosaic virus and barley stripe mosaic virus, but these viruses did not sufficiently infect the plants, and therefore, they could not be used as alternative single guide RNA delivery vectors. In switchgrass, we found that the Cas9-expressing line was unexpectedly resistant to foxtail mosaic virus. We identified a few progeny of the Cas9 line that were susceptible to foxtail mosaic virus, suggesting that genetic resistance is segregating in switchgrass germplasm. However, we were not able to observe genome edits in the switchgrass pds when Cas9 and foxtail mosaic virus expressing the guide RNA were both present. The guide RNA targeting switchgrass pds appeared to be stable in foxtail mosaic virus, unlike the situation in sorghum. We hypothesize that the Cas9 protein an/or the guide RNA delivered by the virus were not accumulating to sufficient levels. We also tested the possibility of using different viruses for guide RNA delivery in switchgrass, but none of the viruses tested were able to sufficiently infect switchgrass. In an alternative strategy, we explored the use of barley stripe mosaic virus for delivery of guide RNAs in barley plants expressing Cas9. The rationale for this approach was that barley stripe mosaic virus was previously shown to induce heritable gene edits in Cas9 wheat plants. It is known that barley stripe mosaic virus is seed transmissible in barley and can also induce transgenerational virus-induced gene silencing in barley, suggesting that this virus is a good candidate to induce heritable gene edits in barley. Under our conditions, we were not able to observe gene editing in barley. However, another group published a paper on heritable gene editing in Cas9 barley using barley stripe mosaic virus to deliver the single guide RNAs. Our hypothesis is that the gene editing may be very condition dependent, and we were unable to replicate the conditions used by the authors of the paper.
Publications
|
Progress 07/01/22 to 06/30/23
Outputs
Target Audience:Researchers interested in using viruses for gene editing or other gene function analysis applications, ag biotechnology industry, and commodity groups. Changes/Problems:In this final year of the project, we will revise Objective 2 to focus on virus-induced gene editing in barley due to the technical hurdles encountered in sorghum and switchgrass. What opportunities for training and professional development has the project provided?A postdoctoral scientist is working on this project, Dr. Melissa Bredow. Dr. Bredow has shared her molecular biology skills with personnel from co-PI labs that have expertise in sorghum (Salas-Fernandez) and switchgrass (Fei). A graduate student from the Fei lab was leading experiments on switchgrass under the supervision of Dr. Bredow has graduated with an MS degree and is now starting his Ph.D. program at the University of Florida. In his new lab, we will continue to collaborate with him on ideas related to virus-induced gene editing in monocot crops. Dr. Bredow has contributed as either co-author or first author on two manuscripts and presented her research at a major international conference. How have the results been disseminated to communities of interest?We published two papers with results that were obtained with partial support from this award. The Bredow et al. 2023 publication describes applications of foxtail mosaic virus for virus-induced gene silencing studies in sorghum. We demonstrate how FoMV VIGS can be used to investigate the functions of genes associated with sorghum defense responses. The Beernink et al. 2023 publication is a comprehensive review of the applications of FoMV for VIGS, gene expression, and gene editing in maize and other monocots. The review provides a history of FoMV vector development, different applications in maize and other plants with a focus on monocots, and experimental design considerations. Dr. Bredow, the postdoctoral scholar working on this project, presented a poster on the characterization of a lipoate protein ligase and its role in the recovery of sorghum photosynthesis following cold stress. This work builds on our previous success in using FoMV for VIGS in sorghum. The poster was presented at the Plant & Animal Genome Conference in San Diego. What do you plan to do during the next reporting period to accomplish the goals?As we have reported previously, there are technical challenges in sorghum and switchgrass that have prevented us from accomplishing the goals of virus-induced gene editing using FoMV or other viruses. Therefore, we have turned our focus on using barley and barley stripe mosaic virus as a system to test virus-induced gene editing. Newly developed Cas9 barley material will be tested for its ability to support virus-induced gene editing. We are ready to start inoculations in the next month and test first for somatic gene editing. If the somatic gene editing is successful, then plants will be maintained and allowed to set seed so that we can test progeny seedlings for the presence of heritable edits in the pds target gene.
Impacts
What was accomplished under these goals?
Impact statement: CRISPR/Cas-based technologies have become important tools for making precise genetic modifications for the improvement of crop plants. However, the delivery of the CRISPR/Cas reagents into plant cells remains a significant bottleneck that limits their utility. Plant viruses have been used to deliver a wide variety of different genetic payloads into plant cells, and most recently, it has been demonstrated that they can deliver CRISPR RNAs or even Cas proteins. The successful delivery of CRISPR/Cas reagents has been demonstrated in most cases in vegetative tissues, such as leaves, and there are a growing number of examples of CRISPR/Cas-induced gene edits that were passed on to progeny through the seed. These inherited gene edits have been demonstrated in the model plants Nicotiana benthamiana and Arabidopsis thaliana, and in the staple crop plant, wheat. We are investigating if similar strategies that work in N. benthamiana, A. thaliana, and wheat can be used to produce virus-induced heritable gene edits in sorghum and switchgrass, which would dramatically accelerate and expand the use of gene editing technologies in these important plant species. While investigating whether we could use FoMV for gene editing in sorghum, we did establish that it is a very effective tool for virus-induced gene silencing (VIGS) that can be used in a wide range of sorghum genotypes for gene function studies. Examples of genes that we have successfully silenced include enzymes that make critical biochemicals, receptor kinases, and an enzyme that functions in the chloroplast, demonstrating that many different pathways and functionalities can be targeted by FoMV VIGS in sorghum. Objective 1: Establish functionality of dicot and monocot FTs in promoting the mobility of Foxtail mosaic virus (FoMV). Work on this objective was completed and published in 2022. Objective 2: Induce heritable gene edits using single gRNAs delivered by FoMV. We have changed focus to investigating gene editing using barley and barley stripe mosaic virus. The past year, we worked with a collaborating lab to generate barley germplasm expressing Cas9 with greater susceptibility to barley stripe mosaic virus. We are now in a position to test our gene editing hypotheses using this system. BSMV clones were constructed that carry different guide RNAs targeting the barley pds gene, and inoculum was generated in N. benthamiana. Objective 3: Enhance editing by FoMV and Sugarcane mosaic virus co-infections. Due to the challenges previously reported in Objective 2, we are no longer pursuing this objective.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Bredow M, Natukunda MI, Beernink BM, Chicowski AS, Salas-Fernandez MG, Whitham SA. Characterization of a foxtail mosaic virus vector for gene silencing and analysis of innate immune responses in Sorghum bicolor. Mol Plant Pathol. 2023 Jan;24(1):71-79. doi: 10.1111/mpp.13270.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Beernink BM, Whitham SA. Foxtail mosaic virus: A tool for gene function analysis in maize and other monocots. Mol Plant Pathol. 2023 Jul;24(7):811-822. doi: 10.1111/mpp.13330.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Bredow M, Panelo J, Kemp J, Whitham, SA, Fernandez MG. Identification and characterization of a lipoate protein ligase gene involved in photosynthesis under cold stress and recovery in sorghum. Plant & Animal Genome Conference 30, San Diego, CA USA. 2023 Jan. Poster presentation.
|
Progress 07/01/21 to 06/30/22
Outputs
Target Audience:Researchers interested in using viruses for gene editing or other gene function analysis applications, ag biotechnology industry, and commodity groups. Changes/Problems:The original proposal was focused on foxtail mosaic virus, but our results suggest that we need to explore the use of other viruses for different technical reasons in sorghum vs switchgrass. Barley stripe mosaic virus was shown to work for heritable guide RNA delivery in wheat, but switchgrass and sorghum lines we are working with are not susceptible to it. We are now attempting to use brome mosaic virus, which is established as a virus-induced gene silencing vectors, as an alternative delivery vehicle. What opportunities for training and professional development has the project provided?A postdoc is working on this project, Dr. Melissa Bredow. Dr. Bredow has shared her molecular biology skills with personnel from co-PI labs that have expertise in sorghum (Salas-Fernandez) and switchgrass (Fei). A graduate student from the Fei lab has been leading experiments on switchgrass under supervision of the postdoc. Dr. Bredow has contributed to writing a manuscript that was published, and is first author on another manuscript that is in review. Dr. Bredow is also mentoring an undergraduate student. How have the results been disseminated to communities of interest?We published one paper with results that were obtained with partial support from this award: Beernink, B. M., Lappe, R. L., Bredow, M., Whitham, S. A. (2022) Impacts of RNA mobility signals on virus induced somatic and heritable gene editing. Front. Genome Ed. 4:925088. doi: 10.3389/fgeed.2022.925088 Whitham gave a presentation on the use of foxtail mosaic virus for gene editing applications to the Iowa State University Crop Bioengineering Center on December 9, 2021.Gene editing in plants using foxtail mosaic virus to deliver guide RNAs. Whitham gave a presentation that included results on the use of foxtail mosaic virus for gene editing applications in the Department of Plant Pathology seminar series at Kansas State University on November 4, 2021. Virus discovery through RNA sequencing and use of viruses in plant gene editing. What do you plan to do during the next reporting period to accomplish the goals?1. Transform new switchgrass lines with a construct to express Cas9, and infect materials derive from these lines for gene editing using FoMV to deliver guide RNAs. 2. Test for somatic edits in sorghum plants infected with BMV carrying guide RNAs fused to different mobility signals. Take plants with somatic edits to seed, and test progeny for heritable edits. 3. Test switchgrass lines for susceptibility to SCMV. 4. Test switchgrass lines for susceptibility to BMV. 5. Test for somatic edits in sorghum Tx430-derived plants with BMV carrying guide RNAs in a co-infection with SCMV.
Impacts
What was accomplished under these goals?
Impact Statement CRISPR/Cas-based technologies have become important tools for making precise genetic modifications for the improvement of crop plants. However, the delivery of the CRISPR/Cas reagents into plant cells remains a significant bottleneck that limits their utility. Plant viruses have been used to deliver a wide variety of different genetic payloads into plant cells, and most recently, it has been demonstrated that they can deliver CRISPR RNAs or even Cas proteins. The successful delivery of CRISPR/Cas reagents has been demonstrated in most cases in vegetative tissues, such as leaves, and there are a growing number of examples of CRISPR/Cas-induced gene edits that were passed on to progeny through the seed. These inherited gene edits have been demonstrated in the model plants, Nicotiana benthamiana and Arabidopsis thaliana, and in the staple crop plant, wheat. We are investigating if similar strategies that work in N. benthamiana, A. thaliana, and wheat can be used to produce virus-induced heritable gene edits in sorghum and switchgrass, which would dramatically accelerate and expand the use of gene editing technologies in these important plant species. Objective 1. Establish functionality of dicot and monocot FTs in promoting mobility of Foxtail mosaic virus (FoMV) RNA. We determined that maize FT homologs ZCN16 and ZCN19 promote gene editing induced by FoMV-based guide RNA delivery. These maize FT homologs performed better than the Arabidopsis FT. However, no FT homolog was able to promote heritable gene edits in Nicotiana benthamiana or maize. Our results indicate that maize FT homologs are expected to perform better in sorghum and switchgrass than the Arabidopsis FT. In addition to FT homologs, we also tested the Arabidopsis tRNA for isoleucine (tRNAIle). tRNAIle was able to also promote gene editing in maize, which suggests to us that it will be useful to test in sorghum and switchgrass. But like the FT homologs, it was not able to promote heritable gene editing events in Nicotiana benthamiana or maize. We also collected data suggesting that RNA mobility signals may not be sufficient to promote heritable gene editing unless the virus has an inherent ability to induce heritable edits when expressing guide RNAs alone. Objective 2. Induce heritable gene edits using single gRNAs delivered by FoMV. Sorghum. We discovered unexpectedly that guide RNAs are very unstable in FoMV in sorghum. This was unexpected, given our previous results with maize. Therefore, we have not proceeded further with guide RNA delivery in sorghum using FoMV. Instead we initiated work to determine if barley stripe mosaic virus (BSMV) can be used as an alternative delivery system, but the sorghum lines that we have expressing Cas9 are not susceptible to BSMV. Subsequently, we have obtained a permit to obtain brome mosaic virus (BMV), which is known to infect sorghum, as another alternative for guide RNA delivery in sorghum. We are now in the process of completing an MTA with Oklahoma State University, which will allow us to bring the BMV system to ISU. The guide RNA instability in FoMV is also surprising, given that we have established that FoMV is a very effective vector for virus-induced gene silencing in sorghum. We silenced two different marker genes and three receptor-like cytoplasmic kinases, and achieved excellent silencing of target genes with consistent and quantifiable loss-of-function phenotypes. This work was a side project that dovetailed with our explorations of the ability of FoMV to infect different sorghum genotypes. We have submitted a manuscript, and it is now under revision. We expect that virus-induced gene silencing with FoMV will become a very useful tool for gene function analyses in sorghum. Switchgrass. To our major disappointment, we discovered that the particular switchgrass clones available to us that express Cas9 are somehow resistant to FoMV. This was unexpected based on preliminary results with wild type switchgrass, and in light of a recent publication by another group, which showed that our FoMV clone is a useful tool for virus-induced gene silencing in switchgrass. Ironically, the wild type switchgrass ecotype is called Alamo, which is the same one from which the Cas9 expressing line was derived. We think that there may be at least two explanations for this resistance. The first is that there could be resistance to FoMV segregating in Alamo, and that we were unlucky that the Cas9 lines were derived from resistant parent material. The second is that the process of tissue culture and regeneration caused a mutation or epigenetic change that rendered the lines resistant to FoMV. Seeds were available from the switchgrass Cas9 lines, and so, we planted them and inoculated with FoMV carrying a guide RNA targeting the two switchgrass phytoene desaturase genes. A few plants were susceptible to FoMV, and one of them also had Cas9. We observed symptoms consistent with photobleaching on this plant, but there we no edits at the target site. We believe that the photobleaching phenotype is due to guide induced gene silencing, which was recently reported. This conclusion is supported by the observation that two other plants that were infected with the FoMV carrying the pds guide RNA did not possess Cas9 but displayed the photobleaching phenotype. It is important to note that switchgrass is self-incompatible, and so, it is an obligate outcrossing plant, which complicates genetic studies. The low frequency of susceptible plants that were segregating combined with segregation of Cas9 means that it is not practical to continue to use these seeds for additional studies. We have tested other switchgrass ecotypes and are now in the process of generating new switchgrass lines expressing Cas9 that are expected to be susceptible to FoMV. Objective 3. Enhance editing by FoMV and Sugarcane mosaic virus co-infections. Due to the challenges described above, we have not made additional progress on this objective beyond what was reported in year 1.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Beernink, B. M., Lappe, R. L., Bredow, M., Whitham, S. A.* (2022) Impacts of RNA mobility signals on virus induced somatic and heritable gene editing. Front. Genome Ed. 4:925088. doi: 10.3389/fgeed.2022.925088
|
Progress 07/01/20 to 06/30/21
Outputs
Target Audience:
Nothing Reported
Changes/Problems:The COVID-19 pandemic caused major delays in hiring as well as staffing levels in my lab due to space restrictions that are now relaxed. I needed to recruit a new postdoc to work on this project who was recently able to join the lab. She is an excellent plant molecular biologist who has been getting up to speed on the viral delivery systems as well as growing sorghum and switchgrass. The pandemic has caused us to get off to a slow start, but we are now in a good position to make rapid progress on the objectives. What opportunities for training and professional development has the project provided?A postdoc was hired and recently began working on this project. The postdoc is sharing her molecular biology skills with personnel from co-PI labs that have expertise in sorghum (Salas-Fernandez) and switchgrass (Fei). 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?1. Conduct experiments to characterize and optimize FoMV infection in switchgrass lines that will be used for gene editing experiments. 2. Test for somatic edits in sorghum plants infected with FoMV carrying guide RNAs fused to different mobility signals. Take plants with somatic edits to seed, and test progeny for heritable edits. 3. Test for somatic edits in switchgrass plants infected with FoMV carrying guide RNAs fusted to different mobility signals. 4. Test switchgrass for suscptibility to SCMV. 5. Test for somatic edits in sorghum Tx430-derived plants with FoMV carrying guide RNAs in a co-infection with SCMV. 6. Test at least one other virus for the possibility of delivering guide RNAs in sorghum and switchgrass.
Impacts
What was accomplished under these goals?
Objective 1. Establish functionality of dicot and monocot FTs in promoting mobility of Foxtail mosaic virus (FoMV) RNA. In a different project that is focused on maize, we found that Arabidopsis FT sequence does not promote mobility of FoMV sufficiently to deliver guide RNAs that can induce heritable edits. This will very likely be the case for sorghum and switchgrass. Therefore, we have already moved to design guide RNAs for sorghum and switchgrass that are fused to an alternative mobility sequence, which is the Arabidopsis tRNA-isoleucine, and we will be focused on monocot FT sequences as opposed to Arabidopsis FT. Objective 2. Induce heritable gene edits using single gRNAs delivered by FoMV. Sorghum: We have established that many sorghum lines are susceptible to FoMV including PI898012 and Tx430, which are the parents of transgenic lines that we are using. Seed from Cas9 sorghum lines in both backgrounds were obtained from on-campus and off-campus collaborators. The off-campus Tx430 Cas9 sorghum was obtained with appropriate APHIS BRS permit, and we also received Tx430 lines that express Cpf1 nuclease as well. Switchgrass: A strategy for preparing Cas9 switchgrass material has been planned, and we are beginning to develop material for inoculation with FoMV and SCMV. Objective 3. Enhance editing by FoMV and Sugarcane mosaic virus co-infections. We established that the Tx430 line is susceptible to sugarcane mosaic virus, but other lines tested, including PI898012, were not susceptible to this virus. Therefore, it will be possible to move forward with this objective with the Tx430-derived lines.
Publications
|
|