Progress 01/15/24 to 01/14/25
Outputs
Target Audience:During this reporting period (Jan 15, 2024 to Jan 15, 2025), we communicated our research to the Agricultural and Environmental Research Staff at Cotton Incorporated. We presented our research as posters and/or oral presentations at several UNT research events. These served to familiarize our undergraduate and graduate students, our postdoctoral scholars and research faculty, and our administrators with the importance of our research on the problems associated with Fusarium wilt disease and aphid infestations in cotton production. We published part of our work in our manuscript "McGarry RC, Lin YT, Kaur H, Higgs H, Arias-Gaguancela O, Ayre BG (2024) Disrupted oxylipin biosynthesis mitigates pathogen infections and pest infestations in cotton (Gossypium hirsutum). J Exp Bot. 10.1093/jxb/erae394". Changes/Problems:There were major changes in project staffing that altered research progress and the rate of expenditures. These were detailed in our third annual report. At that time, PD Ayre and co-PDs McGarry and Shah discussed the remaining budget and the amount of skilled work needed to complete the project. We decided the best course of action was to place co-PD Dr. Roisin McGarry on the project for 90% full-time equivalents. Dr. McGarry supports 90% of her salary from her grants (including this one) and receives 10% salary from UNT's College of Arts and Sciences. We received a no-cost extension through to January 15, 2025, and we anticipated in our third annual report that we would need an additional no-cost extension to recover from these disruptions. What opportunities for training and professional development has the project provided?In Goal 1, Co-PD Dr. Roisin McGarry worked with UNT's Genomics Research Center Manager, Mr. Minh Vu, to sequence 72 transcriptomic libraries; subsequent bioinformatics has been by Dr. McGarry. Co-authors on the published manuscript received training in scientific writing. Goal 2 has been pursued nearly exclusively by a senior graduate student and now postdoctoral scholar (Dr. Yen Tung Lin) as her PhD dissertation. Training has been in tissue culture, microscopy, RNA isolation and gene expression analysis, library construction for transcriptomics, and bioinformatics. Goal 3 has been pursued predominately by Dr. Lin and co-PD Dr. McGarry working extensively with a freshman undergraduate project student, Johnson Chui. Training has been in recombinant DNA technologies, PCR and RT-qPCR, TRV infection for in planta genome editing, DNA and RNA isolation from plants, and high throughput amplicon sequence on an Illumina MiSeq sequencing system. How have the results been disseminated to communities of interest?During this reporting period (Jan 15, 2024 to Jan 15, 2025), we communicated our research to the Agricultural and Environmental Research Staff at Cotton Incorporated. We presented our research as posters and/or oral presentations at several UNT research events. These served to familiarize our undergraduate and graduate students, our postdoctoral scholars and research faculty, and our administrators with the importance of our research on the problems associated with Fusarium wilt disease and aphid infestations in cotton production. We published part of our work in our manuscript "McGarry RC, Lin YT, Kaur H, Higgs H, Arias-Gaguancela O, Ayre BG (2024) Disrupted oxylipin biosynthesis mitigates pathogen infections and pest infestations in cotton (Gossypium hirsutum). J Exp Bot. 10.1093/jxb/erae394". What do you plan to do during the next reporting period to accomplish the goals?We will continue with the research plan described in our original proposal but we acknowledge that we are behind schedule in both research objectives and expenditures. Disruptions to our original graduate student staffing and associated expenditures were detailed in our third annual report. We requested and received a no-cost extension and we also anticipated in our previous project report that a second no-cost extension would be needed to make up for the delays resulting from these staffing changes. Related to Goal 1 but not in the original proposal, we prepared and processed RNA-Seq libraries from control plants, and experimental plants silenced for GhLOX1 and GhLOX5 and infected with FOV. Bio-informatics is underway and we are preparing a manuscript for publication during a no-cost extension. For Goal 2, we are delayed but committed to generating stable GhLOX1 and GhLOX5 knockout edits. Dr. Yen Tung Lin started this work as a PhD student and will continue with this effort while she is still in the laboratory as a postdoctoral scholar. For Goal 3, we will continue to experiment with novel sgRNA mobility factors. Our priority will be to submit a manuscript during a no-cost extension. We had excellent germline editing in N. benthamiana while the same constructs did not promote editing in cotton. Our strategies and results have value to the broader plant biotechnology community.
Impacts
What was accomplished under these goals?
GOAL 1: Activities Completed and Discussion of Results: We demonstrated that the cotton 9-lipoxygenase (9-LOX) encoding genes, GhLOX1, GhLOX5-2, and GhLOX5-9, were induced in response to Fusarium oxysporum f. sp. vasinfectum (FOV), cotton aphids, and Tobacco rattle virus (TRV). Silencing these genes, independently or together, reduced transcripts and metabolites in roots. Importantly, silencing the 9-LOX-encoding genes impaired the progression of FOV and significantly reduced aphid fecundity. Taken together, our findings demonstrated that multiple agents leverage cotton 9-oxylipins in promoting pathogen infections and pest infestations, and reducing the genes encoding these enzymes offers promise to benefit cotton vitality. We submitted a manuscript describing this work. However, reviewers wanted modifications to how we reported our findings and this required repeating experiments. We incorporated an Agrobacterium-only control and confirmed that this treatment did not alter GhLOX1, GhLOX5-2, and GhLOX5-9 expression in roots or shoots. We re-quantified Fusarium wilt progression with closer time points and re-confirmed our initial results among control and experimental plants. In addition to our wilt assay, we monitored disease progression by light microscopy on stem cross sections. Vascular discoloration, a hallmark characteristic of FOV infection, was observed later and was less prevalent in the TRV:GhLOX1 and TRV:GhLOX5 silenced plants compared with uninoculated and empty vector controls, which showed pervasive stem discoloration. We also supported our initial RT-qPCR quantification of GhLOX1, GhLOX5-2, and GhLOX5-9 virus-induced gene silencing (VIGS) with RNA-Seq data. Taken together, our findings demonstrate that multiple agents leverage cotton 9-oxylipins in promoting pathogen infections and pest infestations, and reducing the genes encoding these enzymes offered promise to benefit cotton vitality. To further explore the genetic networks impacted by the interactions of different biotic stresses with fatty acid signaling, we examined the transcriptomes from VIGS plants combined with aphids or FOV challenges. In one experiment, uninoculated, TRV-infected, and TRV:GhLOX1,5-silenced cotton at 21 dpi were treated with FOV or a control for 72 h and RNA was isolated from the roots and systemic leaves from three biological replicates per treatment. In a second experiment, uninoculated, TRV-infected, and TRV:GhLOX1,5-silenced cotton at 21 dpi were treated with cotton aphids or a control for 72 h and RNA was isolated from the leaves bearing the aphid cages and from systemic leaves from three biological replicates per treatment. In total, and in-house, we generated and sequenced 72 transcriptome libraries and analyzed the reads with KBase and other bioinformatics tools, such as ShinyGO. Analysis of this vast dataset continues, but some tantalizing results are emerging. For example, it is evident that aphids and FOV stimulate different genetic networks but have 9-LOX genes and oxylipin signaling in common. Also, FOV infection stimulates numerous genes related oxidative and biotic stress, and silencing 9-LOX-encoding genes further enriches for these gene categories. This is consistent with our model that 9-hydroxy fatty acid oxylipins resulting from 9-LOX activity stimulate many microbes and pests and that targeted disruption of 9-LOX-encoding genes could bolster cotton resilience against prominent threats. Key Outcomes: Cotton 9-LOX-encoding genes, GhLOX1, GhLOX5-2, and GhLOX5-9, are induced in response to FOV, cotton aphids, and TRV. Silencing 9-LOX-encoding genes impairs the progression of FOV and reduces aphid fecundity. This work is published. We generated 72 transcriptomes from VIGS experiments with FOV or aphid challenges to uncover early changes to transcriptional networks in roots and in leaves. Our analyses emphasize that FOV and aphids use different genetic networks in cotton tissues but have 9-LOX-encoding genes in common. GOAL 2: Activities Completed and Discussion of Results: As reported previously, a PhD student generated transgenic cotton with estradiol-inducible expression of embryogenesis and meristem maintenance genes BABYBOOM (BBM), WUSCHEL (WUS), and SHOOT MERISTEMLESS (STM) to potentially improve cotton transformation and regeneration. Also, as reported previously, transcriptomes of WT and transgenic explants cultured with and without WUS induction revealed nearly 4000 and 8000 up- and down-regulated genes, respectively. We designed and constructed Cas9 and Cas12a constructs targeting LOX1 and both LOX5 paralogs. We have not initiated cotton transformation into our WUS-inducible lines, in part because the PhD candidate was focused on bio-informatics and writing and defending her PhD dissertation. She is continuing in the laboratory as a postdoctoral scholar on another project. If a no-cost extension is granted for this award, she has indicated a willingness to continue her former project and create the LOX1 and LOX5 knockout lines. Key Outcomes: Inducing meristem initiation and maintenance gene WUS stimulated somatic embryogenesis and broadly impacts gene expression networks. GOAL 3: Activities Completed and Discussion of Results: A prominent goal is to develop virus-based, meristem gene editing tools to bypass tissue culture and generate heritable, targeted alterations in the cotton germline. As previously reported, we leveraged a 35S:Cas9 transgenic Nicotiana benthamiana (a generous gift from Dr. Daniel Voytas, University of Minnesota) to test potential RNA mobility factors to improve sgRNA delivery into meristem by Tobacco rattle virus (TRV) based vectors: a tRNA sequence, a fragment of Arabidopsis FLOWERING LOCUS T (mAtFT), and sequences from naturally mobile microRNA399 (miR399). We showed that tRNA and FT mobility sequences outperformed miR399 sequencing and we also showed that edits were stably transmitted to the next generation. Our results showed that mAtFT mobility sequences best-promoted germline, heritable editing. In this reporting period, we converted our sgRNA-containing TRV constructs to target G. hirsutum MgChl genes and tested them in cotton using 35:GFP-NLS-Cas9 transgenic Coker 312 (a gift from Dr. Keerti Rathore, Texas A&M). We did not observe white sectors in cotton leaves. Genomic DNA was isolated from each treatment and analyzed for edited genome sequences, but none were detected. We established a collaboration with Dr. Brian Kvitko at the University of Georgia who has a 35S:Cas9-transgenic Jin688 line (a line purported to have better transformation efficiency than Coker 312). In this collaboration, we 1) tested our viruses in this new genetic background; 2) constructed new viruses delivering guide RNAs targeting Ghxa5 (Dr. Kvitko's gene of interest) as well as our own target sequences; and 3) tested our hypothesis that silencing the meristem gene SELF-PRUNING (GhSP) can facilitate access to meristems to impact editing outcomes. We used our well-established methods for TRV delivery. We did not achieve a visual indication of gene editing. We proceeded to analyze the genomic DNA for molecular evidence of editing by amplicon sequencing and the expected edits were not observed. Key Outcomes: Our use of TRV to deliver gRNAs targeting GhMgChl and Ghxa5 did not produce phenotypic or molecular evidence of gene editing in two different transgenic cotton founder lines. Parameters used to successfully edit N. benthamiana did not translate to cotton, but we maintain that in planta meristem editing will be a transformative technology for cotton biotechnology.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
McGarry RC, Lin YT, Kaur H, Higgs H, Arias-Gaguancela O, Ayre BG (2024) Disrupted oxylipin biosynthesis mitigates pathogen infections and pest infestations in cotton (Gossypium hirsutum). J Exp Bot. 10.1093/jxb/erae394
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
McGarry RC, Lin Y-T, Kaur H, Higgs H, Ayre BG (2024) Disrupted oxylipin biosynthesis mitigates pathogen infections and
pest infestations in cotton (Gossypium hirsutum). BioDiscovery Institute Seminar Series, University of North Texas,
Denton, TX. Oral presentation.
|
Progress 01/15/23 to 01/14/24
Outputs
Target Audience:During this reporting period (Jan 2023to Jan 2024), we communicated our research to the Agricultural and Environmental Research Staff at Cotton Incorporated.We presented our research as posters and/or oral presentations at several UNT research events. These served to familiarize our undergraduate and graduate students, our postdoctoral scholars and research faculty, and our administrators with the importance of our research toward the problems associated with Fusarium wilt disease and aphid infestations in cotton production. We similarly presented our research at the annual Cotton Beltwide Conference, where we gave two oral presentations.and at the Plant and Animal Genome Conference, where we gave an oral presentation. These National and International caliber conferences allowed us to network with the cotton research community.Consistent with the subjects of our proposal, these presentations emphasized the role of lipoxygenases in conferring resistance.Stemming from our previously reported provisional patent application to the US Patent and Trademark Office entitled "METHODS AND SYSTEMS FOR TARGETED MODIFICATION OF PLANT MERISTEM GENES AND PLANTS HAVING ENGINEERED MERISTEM MODIFICATIONS", we continued to have video conferences with Bayer Crop Science research scientists. We have a research manuscript submitted for peer review at the Journal of Experimental Botany. Changes/Problems:There have been major changes in project staffing that have altered research progress and the rate of expenditures. Ms. Harmanpreet Kaur was pursuing a Ph.D. degree but life changes necessitated that she leave the project with a Master of Science degree and move to Canada in September 2023. This curtailed the amount of research that she was expected to complete as a PhD student. In September, a new student enrolled in a graduate program to continue this research, but his productivity will be minimal while he takes courses, receives training as a teaching assistant, and learns the techniques and concepts necessary to excel in this project. In addition, another graduate student, Ms. Yen Tung Lin, received support from summer scholarships and preferred to be a teaching assistant supported by the Department of Biological Sciences rather than be a research assistant supported by this grant. This further reduced expenditures from the original budgeted rate. The PD (Ayre) and co-PDs (McGarry and Shah) discussed the remaining budget and the amount of skilled work needed to complete the project. We decided the best course of action was to place co-PD Dr. Roisin McGarry on the project for 90% full time equivalents. Dr. McGarry supports 90% of her salary from her own grants (including this one) and receives 10% salary from UNT's College of Arts and Sciences. The USDA was consulted on this change and we were advised that we could do this internally and that USDA authorization was not needed. We will continue with the research plan described in our original proposal but we acknowledge that we are behind schedule in both research objectives and expenditures. We asked for and received a no-cost extension through to January 15, 2025 and we anticipate that we may also request an additional extension. What opportunities for training and professional development has the project provided?In Goal 1, in addition to the co-PDs of the proposals, two graduate students (Yen Tung Lin and Harmanpreet Kaur, both female), and two undergraduate students (Harrison Higgs and Matthew Feragne, both male) received training and participated in the extensive efforts of tissue harvesting, isolating high-quality RNA, conducting and analyzing the RT-qPCR data, and performing GC-MS analysis for oxylipins. A manuscript is submitted and under review. Goal 2 has been pursued nearly exclusively by a senior graduate student (Yen Tung Lin) as her PhD dissertation. Training has been in tissue culture, microscopy, RNA isolation and gene expression analysis, library construction for transcriptomics, and bioinformatics. Goal 3 has been pursued predominately by co-PD Dr. McGarry working extensively with an undergraduate project student Harrison Higgs. The graduate students and undergraduate students all prepared posters and presented their research at local UNT symposia. Among the undergraduate students, Harrison Higgs and Matthew Feragne were enrolled as a project students and Kiera Allison was supported as a college work/study, hourly laboratory assistant. The graduate students have received some support from this grant, but have also received Teaching Assistantships and summer stipend awards from other sources, and have thus not been supported from this grant to the extent originally budgeted. How have the results been disseminated to communities of interest?During this reporting period (Jan 2023 to Jan 2024), we communicated our research to the Agricultural and Environmental Research Staff at Cotton Incorporated. We presented our research as posters and/or oral presentations at several UNT research events. These served to familiarize our undergraduate and graduate students, our postdoctoral scholars and research faculty, and our administrators with the importance of our research on the problems associated with Fusarium wilt disease and aphid infestations in cotton production. We similarly presented our research at the annual Cotton Beltwide Conference, where we gave two oral presentations. and at the Plant and Animal Genome Conference, where we gave an oral presentation. These National and International caliber conferences allowed us to network with the cotton research community. Consistent with the subjects of our proposal, these presentations emphasized the role of lipoxygenases in conferring resistance. Stemming from our previously reported provisional patent application to the US Patent and Trademark Office entitled "METHODS AND SYSTEMS FOR TARGETED MODIFICATION OF PLANT MERISTEM GENES AND PLANTS HAVING ENGINEERED MERISTEM MODIFICATIONS", we continued to have video conferences with Bayer Crop Science research scientists. We have a research manuscript submitted for peer review at the Journal of Experimental Botany. What do you plan to do during the next reporting period to accomplish the goals?We will continue with the research plan described in our original proposal but we acknowledge that we are behind schedule in both research objectives and expenditures. The graduate students originally budgeted on this grant received summer stipend awards from other sources and also decided to continue with UNT Teaching Assistantships rather than receive Research Assistantships from this grant. This has delayed research progress and also delayed expenditures. We requested and received a no-cost extension to continue this research until January 2025. We anticipate that we may ask for a further extension. Related to Goal 1 but not in the original proposal, we prepared and processed RNA-Seq libraries from control plants, and experimental plants silenced for GhLOX1 and GhLOX5and infected with FOV. Bio-informatics is underway and results will be reported in our next annual report. We are similarly conducting transcriptomic experiments for control plants and experimental plants silenced for GhLOX1 and GhLOX2 and infested with aphids. For Goal 2, the PhD candidate will complete bioinformatics on the RNA-Seq data she generated and she will defend her PhD dissertation. We are committed to generating stable GhLOX1 and GhLOX5 knockout edits, but this will have to be done by someone else. We will likely hire a technician with tissue culture experience to pursue this aspect of the project. For Goal 3, we have obtained a new CRISPR-Cas9 founder line from Christopher Saski, Clemson University, South Carolina. We will repeat our sgRNA delivery with this new line to see if we achieve better results than with our current line.
Impacts
What was accomplished under these goals?
GOAL 1: Activities Completed and Discussion of Results: We previously described results showing that aphid infestations and FOV infections stimulate GhLOX1 and GhLOX5 expression and that our standard methods for reducing gene expression by virus-induced gene silencing (VIGS) attenuated these increases. In this reporting period, we challenged 21-day-old VIGS-treated and control plants with the fungal pathogens, and 72 hours after treatment, VIGS-treated plants with FOV infection showed reduced GhLOX1 and GhLOX5 expression relative to controls. This shows that VIGS reduced target transcripts and that this reduction was maintained when plants were challenged with FOV. We quantified oxylipin species 9-HOT and 9-HOD in the roots of control and VIGS-treated plants 72 hours after FOV infection by HPLC and GC-MS using methods described in our proposal. 9-HOT and 9-HOD oxylipins were detected in control roots that were not silenced with VIGS, but neither were detected in VIGS-treated roots. When challenged with FOV, small quantities were detected in the VIGS-silenced plants. This shows that the VIGS treatment specifically disrupted the biosynthesis of 9-oxylipins and importantly, following FOV infection, metabolite levels were still reduced compared to controls. To test if 9-HOD and 9-HOT abundance affected FOV infection, we monitored the progression of Fusarium wilt disease (i.e., leaf wilting caused by FOV occlusion of the vascular tissues) from 6 to 11 days after infection. VIGS-treated plants showed attenuated symptoms compared with controls. Stem cross-sections were also visualized by microscopy. Six days after infection, vascular discoloration from FOV infection was evident in control plants that did not have LOX genes silenced whereas VIGS-treated plants did not show discoloration at the same time point. Stem discoloration progressed and at 11 days after infection, VIGS-treated plants consistently showed less stem discoloration than controls. Taken together, silencing 9-lipoxygenase genes LOX1 and LOX5 reduced the accumulation of 9-LOX-derived metabolites in roots and this correlated with delayed and attenuated Fusarium wilt disease symptoms. Similar experiments tested if altering the expression of GhLOX1 or GhLOX5 impacted cotton aphid feeding. Aphid infestation increased the expression of GhLOX1 and GhLOX5 in WT plants, as expected, and VIGS treatment partially attenuated this increase but did not completely reverse aphid-induced LOX gene expression. To measure aphid fecundity, cages with five adults were mounted on an expanded leaf of each plant. After 72 h, the leaves were harvested and aphids counted. LOX-silenced, VIGS-treated plants had significantly fewer aphids. During these experiments, we also learned that Tobacco rattle virus (TRV), the virus we use for VIGS, was sufficient to induce GhLOX 1 and GhLOX5 expression. TRV-infected plants (i.e., our empty-vector control) supported larger aphid populations than uninoculated controls, and this is consistent with the elevated expression of the 9-LOX-encoding genes in leaves. Key Outcomes: Transiently silencing the 9-LOX-encoding genes delayed and attenuated the progression of Fusarium wilt disease and reduced cotton aphid fecundity. Our findings support the 9-LOX-encoding genes as promising targets for traditional breeding and biotechnology efforts to bolster cotton resilience against prominent agricultural threats. GOAL 2: Activities Completed and Discussion of Results: As reported in previous reports, a PhD student generated transgenic cotton with estradiol-inducible expression of embryogenesis and meristem maintenance genes BABYBOOM (BBM), WUSCHEL (WUS), and SHOOT MERISTEMLESS (STM) to potentially improve cotton transformation and regeneration. Inducible WUS expression showed the most promise toward enhancing the development of embryonic cultures. In this reporting period, we compared the transcriptomes of WT explants with transgenic explants cultured with and without WUS induction for 7 and 18 days. Nearly 4000 and 8000 genes were up- and down-regulated, respectively, by WUS induction. This shows the broad impacts of WUS and meristem induction on gene expression. Gene ontology analyses categorized many WUS-induced genes as related to cell wall synthesis, photosynthesis, lipid metabolism, and carbohydrate metabolism; this is consistent with the transition of cells to a less differentiated, meristematic state. Furthermore, we determined that WUS induction specifically upregulated transcription factors such as GhWOX13, which is required for callus formation. Our transcriptome analyses suggest that GhWUS induces an embryogenic pathway within somatic cells via the regulation of other transcription factors, such as ABI3 and WOX genes. Supported by the results of Goal 1, we designed and constructed Cas9 and Cas12a constructs targeting LOX1 and both LOX5 paralogs. As of the end of this reporting period, we have not initiated cotton transformation into our WUS-inducible lines, in part because the PhD candidate was focused on bio-informatics and writing her dissertation. Key Outcomes: Inducing meristem initiation and maintenance gene WUS stimulated somatic embryogenesis and broadly impacted gene expression networks. These networks may contain additional targets to improve cotton transformation and regeneration GOAL 3: Activities Completed and Discussion of Results: A prominent goal is to develop meristem gene editing tools to bypass tissue culture and generate heritable, targeted alterations in the cotton germline. As reported in our previous annual report, we leveraged a 35S:Cas9 gene transgenic Nicotiana benthamiana (a generous gift from Dr. Daniel Voytas, University of Minnesota) to optimize our design pipeline. In this reporting period, we continued testing potential RNA mobility factors to improve sgRNA delivery into meristem: a tRNA sequence, a fragment of Arabidopsis FLOWERING LOCUS T (mAtFT), and sequences from naturally mobile microRNA399 (miR399). Our target for germline editing was the N. benthamiana magnesium chelatase (MgChl) subunit I genes. tRNA and mAtFT outperformed sequences from miR399, as evident from extensive white sectors in growing leaves, indicating that MgChl editing was occurring early in organ development. DNA was isolated from white sectors, where all MgChl gene copies were expected to be edited, and from green tissues, where one or more MgChl gene copies were expected to be WT. Sequencing revealed that this was frequently the case, but not always: some white sectors did have WT copies and some green sectors appeared to be completely edited. To test if NbMgChl edits were stably transmitted to the next generation, we examined the phenotypes and genotypes of the M1 generation. Seeds from some flowers produced only green seedlings, while others produced a mixture of green and white (albino) seedlings or only white seedlings. In addition, the edited sequences in the M1 seedlings did not always match the edited sequences in the sector of the parent plant that produced the flower. Taken together, these results suggested that editing was heritable but possibly not clonal. The mAtFT mobility sequences best-promoted germline editing. We converted our sgRNA constructs to target G. hirsutum MgChl genes and tested them in cotton using 35:GFP-NLS-Cas9 transgenic Coker 312 (a gift from Dr. Keerti Rathore, Texas A&M). We did not observe white sectors in cotton leaves. Genomic DNA was isolated from each treatment at the end of this reporting period and these samples are being prepared for amplicon sequencing. Key Outcomes: We confirmed that our strategies for virus-based gene editing yield heritable edits in transgenic N. benthamiana. Attempts to translate this success into cotton have to date been unsuccessful but we maintain that in planta meristem editing will be a transformative technology for cotton biotechnology.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
McGarry RC, Lin Y-T, Kaur H, Higgs H, Ayre BG (2024) Disrupted oxylipin biosynthesis mitigates pathogen infections and pest infestations in cotton (Gossypium hirsutum). BioDiscovery Institute Seminar Series, University of North Texas, Denton, TX. Oral presentation.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
McGarry RC, Lin Y-T, Kaur H, Higgs H, Arias-Gaguancela O, Ayre BG (2024) Disrupted oxylipin biosynthesis mitigates pathogen infections and pest infestations in cotton (Gossypium hirsutum). Plant and Animal Genome Conference / PAG 31, San Diego, CA. Published abstract, displayed poster, and invited oral presentation.
- Type:
Other
Status:
Other
Year Published:
2024
Citation:
McGarry RC, Lin Y-T, Kaur H, Higgs H, Arias-Gaguancela O, Ayre BG (2024) Disrupted oxylipin biosynthesis mitigates pathogen infections and pest infestations in cotton. 2024 Beltwide Cotton Conference, Fort Worth, TX. Published abstract and oral presentations in both the Cotton Improvement Conference and the Cotton Insect Research and Control Conference.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
R�is�n C McGarry, Yen-Tung Lin, Harmanpreet Kaur, Harrison Higgs, Omar Arias-Gaguancela, and Brian G Ayre (202X) Disrupted oxylipin biosynthesis mitigates pathogen infections and pest infestations in cotton (Gossypium hirsutum). Submitted MS ID#: JEXBOT/2024/312757
|
Progress 01/15/22 to 01/14/23
Outputs
Target Audience:During this reporting period (Jan 2022 to Jan 2023), we communicated our research to the Agricultural and Environmental Research Staff at Cotton Incorporated. We had in-person and Zoom research meetings with Dr. Daniel Voytas, an expert in CRISPR-Cas mediated gene editing to discuss our respective strategies to use viruses for in planta editing, from the University of Minnesota Center for Precision Plant Genomics. This has led to a collaboration and exchange of resources. We have his Nicotiana benthamiana Cas9 founder lines, which we are using to initially screen our sgRNA designs and we have provided him with our cotton founder lines. Dr. Roisin McGarry participated in the 2022 Cotton Breeder and Pathology Tour, sponsored by Cotton Incorporated and the Cotton Board. This was an excellent opportunity to exchange ideas and knowledge with cotton professionals from academia, government, non-profit, and industry. As implied in the event name, cotton pathology was a major theme, and aphid pests and Fusarium disease featured prominently. Brian Ayre attended the American Society of Plant Biologists' annual meeting Plant Biology 2022 held in Portland, OR, and discussed gene editing and somatic embryogenesis with collaborators from Israel who were also at the meeting. Dr. Brian Ayre participated virtually in the USDA Agriculture and Food Research Initiative 2022 Critical Agriculture Research and Extension (CARE; A1701) Program Project Director Meeting, held in August 2022, where he presented a poster and a short oral presentation to other attendees. Within the University of North Texas, we presented our work to researchers in the BioDiscovery Institute (BDI) and the BDI External Advisory Board, as well as the Department of Biological Sciences, as part of the broadly attended BioFrontiers weekly seminar series. These presentations reached undergraduate and graduate students with a professional interest in the biological sciences and increase their awareness of research efforts impacting cotton cultivation and production. Stemming from our previously reported provisional patent application to the US Patent and Trademark Office entitled "METHODS AND SYSTEMS FOR TARGETED MODIFICATION OF PLANT MERISTEM GENES AND PLANTS HAVING ENGINEERED MERISTEM MODIFICATIONS", we continue to have video conferences with Bayer Crop Science and BASF Agricultural Solutions research scientists. During this reporting period, COVID-19 restrictions continued to hinder travel to professional conferences. Changes/Problems:There has been a major change in project staffing. Ms. Harmanpreet Kaur was pursuing a Ph.D. degree but a life change necessitates that she move to Canada and leave the project at the end of summer 2023 with a Master of Science degree. This will obviously curtail the amount of research that she will complete toward our project goals. In addition, there are funds budgeted in the grant to support Ms. Kaur as a research assistant which will now be used to support other laboratory members. In the short term (spring and summer of 2023) we have brought in undergraduate seniors to overlap with Ms. Kaur and continue her immediate efforts. For the longer term, it is not clear if a new graduate student will be joining the laboratory in September to continue this project. The PD (Ayre) and co-PDs (McGarry and Shah) are discussing the possibility of combining funds from this project and funds from other projects to hire a technician or (preferably) a postdoctoral scholar to work on similar objectives across multiple projects, such as RNA-Seq and bioinformatics. As of this submission, a decision has not been made. We will continue with the research plan described in our original proposal but we acknowledge that we are behind schedule in both research objectives and expenditures. We will request a no-cost extension. What opportunities for training and professional development has the project provided?In Goal 1, in addition to the co-PDs of the proposals, two graduate students (Yen Tung Lin and Harmanpreet Kaur, both female), two undergraduate students (Harrison Higgs, male, and Kiera Allison, female, African American), and Dr. Guadalupe Lopez-Puc, a visiting scientist from the Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ) in Mexico, have received training and participated in the extensive efforts of tissue harvesting, isolating high-quality RNA, and conducting and analyzing the RT-qPCR data. We are preparing a manuscript. Goal 2 has been pursued nearly exclusively by a senior graduate student (Yen Tung Lin) and outcomes will contribute to her PhD dissertation. Training has been in tissue culture, microscopy, RNA isolation and gene expression analysis, library construction for transcriptomics, and bioinformatics. Goal 3 has been pursued predominately by co-PD Dr. McGarry working extensively with an undergraduate project student Harrison Higgs). Dr. Guadalupe Lopez-Puc, from the Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ) in Mexico, also worked on this project. Among the undergraduate students, Mr. Harrison Higgs was enrolled as a project student and Ms. Kiera Allison was supported as a college work/study, hourly laboratory assistant. The graduate students have received some support from this grant, but have also received Teaching Assistantships and summer stipend awards from other sources, and have thus not been supported from this grant to the extent originally budgeted. How have the results been disseminated to communities of interest?During this reporting period (Jan 2022 to Jan 2023), we communicated our research to the Agricultural and Environmental Research Staff at Cotton Incorporated. We had in-person and Zoom research meetings with Dr. Daniel Voytas, an expert in CRISPR-Cas mediated gene editing to discuss our respective strategies to use viruses for in planta editing, from the University of Minnesota Center for Precision Plant Genomics. This has led to a collaboration and exchange of resources. We have his Nicotiana benthamiana Cas9 founder lines, which we are using to initially screen our sgRNA designs and we have provided him with our cotton founder lines. Dr. Roisin McGarry participated in the 2022 Cotton Breeder and Pathology Tour, sponsored by Cotton Incorporated and the Cotton Board. This was an excellent opportunity to exchange ideas and knowledge with cotton professionals from academia, government, non-profit, and industry. As implied in the event name, cotton pathology was a major theme, and aphid pests and Fusarium disease featured prominently. Brian Ayre attended the American Society of Plant Biologists' annual meeting Plant Biology 2022 held in Portland, OR, and discussed gene editing and somatic embryogenesis with collaborators from Israel who were also at the meeting. Dr. Brian Ayre participated virtually in the USDA Agriculture and Food Research Initiative 2022 Critical Agriculture Research and Extension (CARE; A1701) Program Project Director Meeting, held in August 2022, where he presented a poster and a short oral presentation to other attendees. Within the University of North Texas, we presented our work to researchers in the BioDiscovery Institute (BDI) and the BDI External Advisory Board, as well as the Department of Biological Sciences, as part of the broadly attended BioFrontiers weekly seminar series. These presentations reached undergraduate and graduate students with a professional interest in the biological sciences and increase their awareness of research efforts impacting cotton cultivation and production. Stemming from our previously reported patent application to the US Patent and Trademark Office entitled "METHODS AND SYSTEMS FOR TARGETED MODIFICATION OF PLANT MERISTEM GENES AND PLANTS HAVING ENGINEERED MERISTEM MODIFICATIONS" (application number 17/749,560), we continue to have video conferences with Bayer Crop Science and BASF Agricultural Solutions research scientists. During this reporting period, COVID-19 restrictions continued to hinder travel to professional conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue with the research plan described in our original proposal but we acknowledge that we are behind schedule in both research objectives and expenditures. We will request a no-cost extension. For Goal 1, we are repeating the experiments described to strengthen our current results. Metabolite analyses to quantify levels of oxylipins were initiated after the close of this reporting window, and a priority for this next reporting window will be to continue those efforts. We will complete our manuscript currently in preparation. For Goal 2, the graduate student working on this aspect of the project will complete bioinformatics on the RNA-Seq data she generated and she will complete and defend her PhD dissertation. She will use the WUS-inducible lines she generated and her new CRISPR-Cas9 and Cas12a constructs to make stable GhLOX1 and GhLOX5 knockout edits. For Goal 3, we will continue to use N. benthamiana to quickly screen viral constructs for sgRNA delivery and we will test the most promising constructs in cotton to generate GhLOX1 and GhLOX5 knockout edits in planta without tissue culture. We will complete our manuscript currently in preparation.
Impacts
What was accomplished under these goals?
Impact: Cotton (Gossypium hirsutum) is our most important fiber crop. Yield and quality are undermined by pests and pathogens, including cotton aphids and Fusarium oxysporum pv. vasinfectum (FOV) fungal infections. Plant-synthesized oxylipins, specifically 9-hydroxy fatty acids resulting from 9-lipoxygenase activity (9-LOX), are feeding stimulants for many pests, and reduced levels provide protection. Addressing Critical Agricultural Research and Extension (A1701), we propose to develop genome editing approaches to confer natural resistance against cotton aphid and Fusarium wilt by elucidating oxylipin signaling and manipulating its derivatives. GOAL 1: Activities Completed and Discussion of Results: We continued the analysis initiated and reported in our first annual report. Cotton plants with and without FOV infection and with and without aphid infestation were established for a time-course experiment. At T=24, 72, and 144 hr, tissue was isolated from the root, mature leaf, and shoot apex and stored at -80 °C. RT-qPCR was used to quantify GhLOX1, GhLOX5-2, and GhLOX5-9 transcripts, and heat maps were used to visualize changes in transcript abundance. All three genes were upregulated in the presence of FOV and aphids in roots with a lesser but still significant increase in shoots. We used Tobacco rattle virus (TRV) and our standard protocols for VIGS, we demonstrated that we could silence GhLOX1 alone (construct "TRV-LOX1"), both GhLOX5 paralogs together (construct "TRV-LOX5"), as well as all three genes together ("TRV-LOX1,5") throughout the plants. Importantly, silenced plants showed no physiological or developmental differences compared with controls. We tested if LOX-silenced plants were more resilient to aphids and FOV by challenging three-week-old VIGS-treated plants with either aphid or with FOV. Effective VIGS treatment was confirmed by RT-qPCR, but in the respective experiments, aphid counts were highly variable among replicate plants and the impact of VIGS on FOV progression was inconclusive. However, the VIGS-treated plants did show continued silencing throughout the experiment. With what we learned from these efforts, we have refined and we will repeat these experiments. Key outcomes: We demonstrated that FOV infections and cotton aphid infestations induce GhLOX1 and GhLOX5 expression. We demonstrated that VIGS reduces GhLOX1 and GhLOX5 expression and that these reductions are maintained during FOV and aphid treatments. Silencing GhLOX1 and GhLOX5 expression did not result in an obvious reduction of FOV infection or cotton aphid infestation under the conditions tested, but we have designed refined experiments to test for a more nuanced enhancement of resistance. GOAL 2: Activities Completed and Discussion of Results: As a means to potentially improve cotton transformation and regeneration, a graduate student generated transgenic cotton in the Coker 312 background with inducible expression of embryogenesis and meristem maintenance genes LEAFY COTYLEDON 2, BABYBOOM, WUSCHEL (WUS), and SHOOT MERISTEMLESS. In each, the experimental gene is induced by 17β-estradiol, and effectiveness was assessed by visual observation for embryogenesis as well as RT-qPCR to quantify gene induction. Several lines with inducible WUS expression showed the most promise. High levels of WUS induction were deemed toxic because tissue quickly turned brown and died. Two lines with "moderate" induction were used to optimize both the concentration of inducer (10 μM) and the length of time for exposure (72 hr) and these explants appear to have improved embryogenesis and subsequent regeneration potential compared to untransformed controls subjected to the same conditions. As of the end of this reporting period, the graduate student is repeating these experiments to better quantify these outcomes. To gain insight into the molecular events contributing to WUS-induced embryogenesis and meristem formation, we performed a time course (T=24 hr, 72 hr, and 144 hr) on induced and uninduced explants and performed RNA-Seq transcriptomics. Bioinformatics on these large data sets is ongoing as of the end of this reporting period. We hypothesize that this information will give important insight into the genetic networks that are activated by WUS induction and will also determine if there are expected genes and networks that are not being activated; this will ultimately inform on future targets for improved embryogenesis and regeneration in cotton. As indicated in our first annual report, we did not initiate efforts to use Cas9 and Cas12a to create stable mutations in cotton LOX genes until the impact of LOX1 and LOX5 loss-of-function is better defined through our efforts with VIGS in Goal 1. With the experiments and outcomes described for Goal 1 above, we designed Cas9 and Cas12a constructs targeting LOX1 and both LOX5 paralogs. As of the end of this reporting period, sgRNA components Cas9 and Cas12a backbones have been ordered from third-party vendors. These constructs will be transformed into our WUS-inducible lines. Key outcomes: A graduate student established transgenic cotton lines with inducible WUS expression. These have been propagated through the F2 generation and seeds homozygous for the transgene are identified. These lines establish a foundation for future experiments both for this project (see Goal 3) and also, potentially, as a resource for the broader cotton community. GOAL 3: Activities Completed and Discussion of Results: We previously reported that using VIGS to silence GhSP (cotton SELF-PRUNING) causes a dramatic transition to reproductive growth and enhances the penetrance of virus-based tools into the meristematic zone. We hypothesize that co-silencing GhSP while delivering sgRNA will facilitate creating edited sectors that lead to edited flowers and ultimately edited seeds. As reported in our first annual report, we achieved editing but at a frequency too low to be of practical use. During this reporting period, we decided we needed a more efficient system to test our sgRNA delivery constructs. We hypothesized that naturally mobile microRNAs and other mobility sequences could promote viral delivery of sgRNAs to the meristem. Recombinant TRV viruses were introduced to Nicotiana benthamiana plants expressing a Cas9 transgene. Infected plants showed sectoring in leaves soon after inoculation, suggesting early editing of target genes. While the extent of sectoring varied among virus treatments, viral RNA was detected in the systemic leaves of all inoculated plants. Genomic DNA was isolated from the first and seventh systemic leaves, and from progeny seedlings, and analyzed by amplicon sequencing. We found that the mobility factors tested differentially affected somatic and heritable gene editing. Sequences derived from FLOWERING LOCUS T (FT) and tRNA were more effective than our microRNA designs. We are refining our constructs and we will test this in cotton to see if results from N. benthamiana can be translated to cotton and perhaps other crops. Key outcomes: We demonstrated that have sgRNA designs that very effectively create heritable edits in N. benthamiana. This is not novel, since effective virus-based gene editing has been shown previously in N. benthamiana but importantly, we demonstrate that we have a method to quickly screen experimental designs and this will allow us to more efficiently focus on optimized designs for translation to cotton plants.
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Progress 01/15/21 to 01/14/22
Outputs
Target Audience:During this reporting period (Jan 2021 to Jan 2022), we communicated our research to Agricultural and Environmental Research Staff at Cotton Incorporated. We had one-to-one research meetings by Zoom with research scientists at Auburn University in Alabama to discuss cotton aphid entomology and to establish our own cotton aphid colony at UNT. We had Zoom and in-person research meetings with scientists from Texas A&M University to discuss cotton transformation, cotton gene editing, virus-based gene manipulation strategies for cotton, and to establish our Fusarium oxysporum f. sp. vasinfectum Race 4 (FOV4) culture. We also had in-person discussions with research scientists from the Center for Precision Plant Genomics at the University of Minnesota to discuss cotton transformation, gene editing, and virus-based gene-editing strategies. Within the University of North Texas, we presented our work to researchers in the BioDiscovery Institute (BDI) and the BDI External Advisory Board. Stemming from research related to this project, we submitted an intellectual property disclosure in the form of a provisional patent application to the US Patent and Trademark Office entitled "METHODS AND SYSTEMS FOR TARGETED MODIFICATION OF PLANT MERISTEM GENES AND PLANTS HAVING ENGINEERED MERISTEM MODIFICATIONS". Based on this IP, we had video conferences with research scientists at Bayer Crop Science and BASF Agricultural Solutions. Our research has not yet progressed to having publication-ready results, and during this reporting period, COVID-19 restrictions continued to hinder travel to professional conferences. Changes/Problems:There are no "major" changes or problems to report. As described above, we have changed the Fusarium line that we are using to one that is much more relevant to cotton production. What opportunities for training and professional development has the project provided?Two graduate students, both pursuing PhDs, were supported as research assistants (RAs) on this project (Harmanpreet Kaur and Yen-Tung Lin, both female). These graduate students, more or less equally, completed most of the work described in this project report, under the supervision of the project directors. Consistent with UNT policy and our approved budget, this RA support included tuition costs. One undergraduate student (Kiera Allison, female, African American) was supported as hourly technical support for about 10 to 12 hours per week. Training included general wet-lab tasks, such as preparing media and solutions, cleaning glassware, autoclaving, and plant maintenance, including sterile tissue culture. This undergraduate student also had College Work-Study support, so this USDA grant supported ~1/3 of her wages. One visiting research professional, Dr. Guadalupe Lopez-Puc, who is on sabbatical leave at UNT for 12 months from Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ), Plant Biotechnology Unit, participated in aphid infestations and FOV infection, as well as harvesting tissues and RNA isolation. This participation was not supported financially by the grant. How have the results been disseminated to communities of interest?During this reporting period (Jan 2021 to Jan 2022), we communicated our research to Agricultural and Environmental Research Staff at Cotton Incorporated. We had one-to-one research meetings by Zoom with research scientists at Auburn University in Alabama to discuss cotton aphid entomology and to establish our own cotton aphid colony at UNT. We had Zoom and in-person research meetings with scientists from Texas A&M University to discuss cotton transformation, cotton gene editing, virus-based gene manipulation strategies for cotton, and to establish ourFusarium oxysporumf. sp.vasinfectumRace 4 (FOV4) culture. We also had in-person discussions with research scientists from the Center for Precision Plant Genomics at the University of Minnesota to discuss cotton transformation, gene editing, and virus-based gene-editing strategies. Within the University of North Texas, we presented our work to researchers in the BioDiscovery Institute (BDI) and the BDI External Advisory Board. Stemming from research related to this project, we submitted an intellectual property disclosure in the form of a provisional patent application to the US Patent and Trademark Office entitled "METHODS AND SYSTEMS FOR TARGETED MODIFICATION OF PLANT MERISTEM GENES AND PLANTS HAVING ENGINEERED MERISTEM MODIFICATIONS". Based on this IP, we had video conferences with research scientists at Bayer Crop Science and BASF Agricultural Solutions. Our research has not yet progressed to having publication-ready results, and during this reporting period, COVID-19 restrictions continued to hinder travel to professional conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue with the research plan described in our original proposal. Our immediate goals are to combine LOX1 and LOX5 silencing with aphid infestations and FOV4 infection to assess the impact of LOX1 and LOX5 loss of function on aphid infestations and FOV4 infection, as described in Objective 1. We will also pursue metabolomic and transcriptomic analysis as described in Objective 1 of the proposal. Testing new constructs and new targets for virus-based gene editing, as described in Objective 3, is a priority.
Impacts
What was accomplished under these goals?
During this reporting period, we established a colony cotton aphid (Aphis gossypii) with the help of Dr. Alana Jacobson at Auburn University. We also established a culture of Fusarium oxysporum f. sp. vasinfectum Race 4 (FOV4) with the help f Dr. Libo Shan at Texas A&M University (Cox et al., 2019; Wang et al., 2020). This isolate of Fusarium is much more relevant to cotton production than Fusarium graminearum, which was proposed in the original grant submission. We are no longer working with F. graminearum, and all future work will be with FOV4. Towards the goals outlined in Objective 1, we established a time course for both FOV4 infection and for aphid infestation in 4-week old plants. Samples were collected from root, stem, and leaves of plants that were infected with FOV4 or infested with aphids, as well as uninfected and uninfested control plants after 24, 72, and 144 hours. Samples were collected into liquid nitrogen and stored at -80 °C. RNA was isolated by a CTAB procedure (Gambino et al., 2008), residual DNA in the samples was removed by DNase treatment. The 9-Lox lipoxygenases hypothesized to create the oxylipin compounds proposed to stimulate Fusarium infection and aphid feeding are encoded by LOX1 and LOX5 genes. LOX1 and LOX5 gene expression levels were quantified by RT-qPCR. Consistent with expectations and published results from other systems (Nalam et al., 2012), Fusarium infection increased LOX1 expression in roots within 24 hours and LOX5 expression in roots within 72 hours. Aphid infestation increased LOX5 expression in roots within 24 hours for an allele on chromosome 2, and within 72 hours for an allele on chromosome 9. Expression of both alleles dropped to below levels observed in control plants by 144 hours. Expression levels of LOX1 were not affected by aphid infestation. The results need to be confirmed by repeating the experiment. In addition, we constructed Tobacco rattle virus (TRV) constructs for virus-induced gene silencing (VIGS) (McGarry et al., 2017; McGarry et al., 2020) of LOX1 and LOX5 alleles. Our TRV VIGS constructs were inoculated into 4-day old cotyledons and root, shoot, and leaf samples were isolated into liquid nitrogen after 21 days and stored at -80 °C. RNA was isolated and DNA digested with DNase. RT-qPCR analysis showed silencing of the target gene by up to 50% in most organs. The results need to be confirmed by repeating the experiment. We are currently combining LOX1 and LOX5 silencing with aphid infestations and FOV4 infection to assess the impact of LOX1 and LOX5 loss of function on aphid infestations and FOV4 infection. Objective 2 involves creating transgenic plants with CRISPR-mediate edits in LOX1 and LOX5 genes. We will not initiate these efforts until the impact of LOX1 and LOX5 loss of function is better defined through our efforts with VIGS in Objective 1. Toward Objective 3, we tested our Tobacco rattle virus (TRV) vectors and our Cotton leaf crumple virus (CLCrV) vectors (McGarry et al., 2017; McGarry et al., 2020) carrying sgRNA targeting MAGNESIUM CHELATASE subunit 1 (MgChl1) in our CRISPR-Cas9 line obtained from Dr. Keerti Rathore, Texas A&M University. The sgRNA was expressed from an AtU6 promoter, and effectiveness of the sgRNA alone was compared to sgRNA fused to a truncated sequence of FLOWERING LOCUS T (FT), which others have reported to improve systemic movement (Ellison et al., 2020; Maher et al., 2020). We also tested the effectiveness of the sgRNA constructs alone, and co-infection with TRV-GhSP, which causes dramatic termination of vegetative growth and the transition to reproductive growth at all meristems (McGarry et al., 2016; McGarry et al., 2020). Our own (unpublished) results from other projects argue that GhSP silencing enhances penetrance of virus-based tools into the meristematic zone, and as described in our proposal, we hypothesize that co-silencing GhSP while delivering sgRNA facilitate creating edited sectors that lead to edited flowers and ultimately seeds. Both three weeks and ten weeks post inoculation, 96 samples were collected from inoculated cotyledons and from various systemic organs, including leaves, apical buds, and flower buds. DNA was isolated and the target regions were barcoded and amplified by PCR before amplicon sequencing on an Illumina MiSeq device (Xue and Tsai, 2015). This approach resulted in ~10,000 reads for each of the 96 samples. In the inoculated cotyledons, approximately 1% of the sequences showed desired edits in the target sequence. In systemic tissues, the frequency of editing was approximately 10-fold less. Thus, targeted gene editing occurred - which is a positive outcome - but the frequency and efficiency of editing in these experiments was disappointing. New experiments with new targets and new constructs have been designed to continue the research outlined in Objective 3 and to improve in planta editing efficiency. Reference Cox KL, Babilonia K, Wheeler T, He P, Shan LB (2019) Return of old foes - recurrence of bacterial blight and Fusarium wilt of cotton. Current Opinion in Plant Biology 50: 95-103 Ellison EE, Nagalakshmi U, Gamo ME, Huang P-j, Dinesh-Kumar S, Voytas DF (2020) Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs. Nature Plants 6: 620-624 Gambino G, Perrone I, Gribaudo I (2008) A Rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants. Phytochem Anal 19: 520-525 Maher MF, Nasti RA, Vollbrecht M, Starker CG, Clark MD, Voytas DF (2020) Plant gene editing through de novo induction of meristems. Nature Biotechnology 38: 84-89 McGarry RC, Klocko AL, Pang M, Strauss SH, Ayre BG (2017) Virus-Induced Flowering: An Application of Reproductive Biology to Benefit Plant Research and Breeding. Plant Physiology 173: 47-55 McGarry RC, Prewitt SF, Culpepper S, Eshed Y, Lifschitz E, Ayre BG (2016) Monopodial and sympodial branching architecture in cotton is differentially regulated by the Gossypium hirsutum SINGLE FLOWER TRUSS and SELF-PRUNING orthologs. New Phytologist 212: 244-258 McGarry RC, Rao X, Li Q, van der Knaap E, Ayre BG (2020) SINGLE FLOWER TRUSS and SELF-PRUNING signal developmental and metabolic networks to guide cotton architectures. J Exp Bot 71: 5911-5923 Nalam VJ, Keeretaweep J, Sarowar S, Shah J (2012) Root-Derived Oxylipins Promote Green Peach Aphid Performance on Arabidopsis Foliage. The Plant Cell 24: 1643-1653 Wang P, Zhou L, Jamieson P, Zhang L, Zhao ZX, Babilonia K, Shao WY, Wu LZ, Mustafa R, Amin I, Diomaiuti A, Pontiggia D, Ferrari S, Hou YX, He P, Shan LB (2020) The Cotton Wall-Associated Kinase GhWAK7A Mediates Responses to Fungal Wilt Pathogens by Complexing with the Chitin Sensory Receptors. Plant Cell 32: 3978-4001 Xue LJ, Tsai CJ (2015) AGEseq: Analysis of Genome Editing by Sequencing. Molecular Plant 8: 1428-1430
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