FUNCTIONAL VALIDATION OF CANDIDATE GENES IN HOPS THROUGH CRISPR/CAS9 MUTAGENESIS SYSTEMS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1026174
• Grant No.: 2021-67013-34739
• Cumulative Award Amt.: $291,000.00
• Proposal No.: 2020-05677
• Project Start Date: Jul 1, 2021
• Project End Date: Jun 30, 2024
• Grant Year: 2021
• Program Code: [A1191]- Agricultural Innovation through Gene Editing

Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331

Performing Department
Forest Ecosyst & Society

Non Technical Summary
Hops are specialty crop in the US prized for their value in brewing beer. Due to the explosive growth of the craft beer market throughout the last decade, demand for hops and overall production have been steadily increasing. Although once a regional crop grown almost exclusively in the Pacific Northwest, other minor production areas are emerging throughout the US as interest in locally grown agricultural products has risen. Plant diseases are a major challenge faced by hop growers. Powdery mildew, a fungus, is particularly problematic in the major hop growing areas. Millions of dollars in revenue are lost annually to decreased hopyard productivity and increased labor costs due to management of this disease. Pesticide applications and practices such as early pruning can help limit the spread of powdery mildew, but the most reliable way of controlling the disease is by planting hop varieties that have built-in genetic resistance to it. Thus, powdery mildew resistance is a highly prioritized trait in breeding of new hop varieties. The best-studied source of genetic resistance to powdery mildew strains prevalent in the Pacific Northwest is referred to as R6. The exact gene that causes R6 resistance is not known, but previous research using genetic mapping has determined its location to be within a small region of one of the chromosomes in the hop genome.Genetic engineering could be a very useful tool in narrowing down the identity of the gene that causes R6 resistance in hop. However, no procedures for genetic engineering of American hop varieties, including ones that have the R6 trait, have been published in the scientific literature up to this point. Therefore, the first objective of this project is to develop a procedure for genetic engineering in an American hop cultivar by testing several hop varieties while adjusting multiple different variables in the genetic engineering process. One new technique within genetic engineering, referred to as gene editing, uses a set of molecular "machinery" called CRISPR/Cas9 to turn a specific gene on, off or otherwise alter it by making minor changes to its DNA sequence. For the second objective, we plan to use CRISPR/Cas9 to turn off several genes within the region where the R6 gene is known to be located in the hop genome. The third objective involves exposing plants that have been edited in this way to the powdery mildew fungus, which will allow us to determine the specific gene responsible for R6. The fourth and final objective involves exploring a technique that will help to develop gene edited plants in which the CRSIPR/Cas9 machinery has been removed from the plant's cells after it has already done its editing job.If this project is successful, it will help to improve hop breeding for powdery mildew resistance which would lead to reduced expenses for hop growers and ultimately for brewers and beer consumers. Furthermore, the procedures developed here would pave the way for future research in hop genetics and would open a route for gene editing to be used directly as a breeding technique to more quickly improve hop cultivars.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	2230	1080	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
2230 - Hops;

Field Of Science
1080 - Genetics;

Keywords

crispr

hops

humulus

biotechnology

genetic engineering

genome

powdery mildew

transformation

Goals / Objectives
Goal 1- Establish a genetic transformation system for a USA hop cultivar while simultaneously generating gene-edited hop plants to study the basis of an important agronomic trait.Build multiple binary plasmid constructs carrying CRISPR/Cas9 editing reagents designed for targeting each of several candidate R genes potentially responsible for conferring a major source of resistance to hop powdery mildew (PM)Test multiple hop cultivars for tissue culture regeneration response as well as capacity for transient Agrobacterium-mediated transformation, measuring the latter using the expression of GFP contained in the designed plasmid constructs.For 1-2 top-performing cultivars, optimize regeneration/transformation efficiency by varying tissue cultures parameters and transformation techniques.Identify transgenic plants carrying mutant alleles for each of the target candidate genes through sequencing.Phenotype bi-allelic mutant plants for each candidate gene by challenging leaves with an appropriate strain of the powdery mildew pathogen Podosphaera macularisGoal 2 - Test and apply a method for obtaining CRISPR-edited hop plants that are transgene-free.Build a CRISPR plasmid construct for targeting the phytoene desaturase (PDS) gene in hopTransform the cultivar of choice with the CRISPR-pds construct using the previously optimized transformation protocol.Using the chlorotic/albino phenotype produced by PDS inactivation and the expression of GFP as visual markers of CRISPR editing and transgene integration, respectively, test the effect of various gradations of "relaxed selection" during regeneration on the recovery rate of edited, transgene-free de novo shootsUse PCR to confirm the absence of CRISPR reagents in genomic DNA of mutant plants and perform Sanger sequencing of pds alleles to validate editing

Project Methods
The methods outlined here will test three hypotheses:A hop transformation system with a transgenic plant recovery rate comparable to or higher than those of previously established protocols can be developed for an important USA cultivar through evaluation of various genotypes, tissue culture amendments, and ancillary treatments.At least one of the three predicted R genes within the putative R6 locus in the latest hop genome assembly that were highly expressed in leaf tissue according to RNA-seq data (annotated as genes 44, 49, and 53 on contig 000559F) is responsible for conferring R6 resistance to hop powdery mildew, and not the three other weakly expressed genes at that locus.By manipulating the timing and duration of antibiotic selection in tissue culture media, we can substantially increase the proportion of transiently edited plants out of all regenerated shoots, relative to the proportion obtained when no selection is used. By the combined use of GFP and pds reporters, we can do this efficiently and at a large enough scale to produce robust data.In developing and optimizing the transformation system, the following methods will be used:Test the regeneration response of several US hop cultivars alongside the European landraces used in previously established transformation protocols (as positive controls) with varying tissue culture conditions such as indirect vs. direct regeneration protocols, types/concentrations of auxins and cytokinins in regeneration media formulations, and susceptibility to different antibiotics that could be used for tissue selectionTest the transient transformation efficiency of these same cultivars through varying parameters such as Agrobacterium strain used for gene transfer, pre-induction with acetosyringone, and treatment with a surfactant during Agrobacterium inoculation.Optimization for the above two experiments will be evaluated by collecting data on the percentage of explants forming green organogenic callus on shoot induction medium up to 4 weeks after transfer and the extent of GFP expression on explants/callus one week after transfer from co-cultivation, respectively.After 1-2 top-performing hop genotype/Agrobacterium strain combinations are chosen with optimal regeneration process, hormone treatment, and antibiotic selection parameters based on previous experiments, the transformation protocol will be further optimized by testing if altering Agrobacterium inoculum density, adding antioxidant compounds to co-cultivation medium, or use of methods such as tissue sonication, wounding, or vacuum infiltration can further boost transformation efficiency.Stable transformation efficiency will be evaluated by counting the number of GFP-expressing de novo shoot buds obtained 8-12 weeks following transformation. The final transformation protocol will be considered comparably efficient to previously developed hop transformation protocols if stable transformation rates of 3-5% or higher can be achieved.For functional testing of the candidate R genes, the following methods will be used:A Golden Gate cloning kit will be used to generate individual plasmids for targeting each of the candidate R genes for knockout and these will be mobilized to several different Agrobacterium strains. Two Agrobacterium cultures of the same strain carrying plasmids for targeting two distinct R genes will be mixed together in equal proportions prior to transformation to reduce screening workload. These pooled Agrobacterium inocula will be used in the previously outlined transient and stable transformation experiments.Transgenic plants resulting from the protocol development and optimization experiments will be carried forward in tissue culture at least until they reach a stage where leaf tissue can be sampled for genotyping.Alleles for the R genes targeted by the specific Agrobacterium pool used to inoculate each set of transgenic plants will be PCR amplified and genotyped by Sanger sequencing.Additional transformation using the optimized protocol will be performed as needed to acquire the requisite number of edited plants for phenotyping experiments.Plants confirmed to be bi-allelic mutants for a given candidate gene will be brought to maturity and propagated. Unfurled leaves of these plants will then be inoculated in a contained culture with strains of Podosphaera macularis and compared to the infection of wild-type leaves. A qualitative susceptibility response in contrast to the resistance in the wild-type genetic background is expected to result from the knockout of at least one of the candidate genes.In evaluating the relaxed selection strategy for obtaining transgene-free edited plants, the following methods will be used:A plasmid construct targeting the single-copy gene PDS, which will have been designed in parallel to the other CRISPR constructs, will be used to transform hop plants using the optimized protocol.As transformed explants incubate on regeneration media, varying concentrations and duration of exposure to the selective agent will be applied through modification of media formulations to administer fractions of the full concentration required to obtain only stable transgenic plants in the developed protocol.Using the visually detectable albino phenotype induced by pds and GFP expression as markers of successful editing and transgene presence, respectively, the proportion of edited, transgene-free plants in relation to the total number of explants receiving a certain relaxed selection treatment will be evaluated.If the rate of clean-edited plant recovery is substantially higher using a relaxed selection scheme than the control condition in which no selection is applied, then this may be a viable strategy for producing transgene-free CRISPR mutants for which no early marker is evident following knockout of the target gene.PCR amplification with primers specific to the transgene will be used to confirm the absence of an integrated transgene copy in mutant plants and Sanger sequencing will be used to characterize the mutations made within the PDS target gene.Quantitative analysis. To enable statistical analysis of genetic effects, we will produce at least five independent transgene insertion events that lead to bi-allelic knockouts of each target gene as well as Cas9-only control events. All regeneration and transgene expression experiments will employ a minimum of three replicates consisting of separate Petri dishes per treatment, with about 20 explants per plate. Sample sizes will be increased to 4-5 replicates for tests of stable transformation efficiency and transient gene editing. For disease phenotyping experiments, replicates consisting of multiple ramets from each bi-allelic mutant (transgenic event) will be used to derive leaves for P. macularis inoculation. Mixed model ANOVA will be used to test measures such as percentage of explants forming callus, number of shoots regenerated per explant per plate, number of calli or regenerating shoots showing GFP expression (or chlorotic pds phenotype) per plate, and disease severity score in leaves derived from the same transgenic event within the population of transformants mutant for multiple R genes. Trait transformations and non-parametric analyses will be employed as needed to satisfy model assumptions.

Progress 07/01/21 to 06/30/24

Outputs
Target Audience:Our target audiences we have engaged with during this project included stakeholders in the hop growing and brewing industries through poster and speaking presentations at multiple regional and national conferences as well as through one-on-one meetings with industry representatives. Lead PI Strauss has attended the 2022 Brewing Summit in Providence, RI (hosted jointly by the Master Brewers Association of the Americas and the American Society of Brewing Chemists). There, he was invited to give an oral presentation on the implications of biotechnology in hop as it relates to brewing, as well as to participate in a panel discussion with other researchers involved in biotechnology with respect to other brewing ingredients. We also presented a poster at this conference, which communicated our project's progress and goals, and potential utility in non-technical language for the purpose of disseminating our research to the largely brewing industry audience in attendance. Dr. Strauss also participated in a similar session at the 2023 Master Brewers Conference in Seattle, WA, October 6-8. He was also invited to participate in an episode of the Master Brewers Podcast to discuss our research and the larger scope of hop biotechnology in brewing. Key personnel (Willig) has presented updates on the project research at the Washington Hop Commission and the Hop Research Council annual and semi-annual meetings (respectively) for audiences of hop growers, merchants, and brewing representatives. Willig has also partnered with other yeast and barley researchers to prepare panel discussions on biotechnology in brewing for two different brewing conferences. Finally, Willig has been a guest on two different brewing-related podcasts to disseminate results and discuss implications of the work on this project to the wider brewing community. This project has led to training of three undergraduate researchers including both hands-on laboratory training and informal instruction in plant transformation and molecular biology theory. One of these student researchers has been specifically designated to work on this project for about 75% of the total period. Changes/Problems:As discussed in both of the progress reports, we encountered difficulties which limited the feasibility of characterizing the R6 candidate genes we originally intended to target for gene editing. The hop varieties that were amenable to regeneration and transformation in our experiments were not ones that display the R6 dominant phenotype. Furthermore, hop genomic sequences assembled since the start of the project showed the R6 candidate locus to be highly variable and to display copy number variation across genotypes, making the sequence(s) that should be targeted uncertain without production of additional sequencing. We had the opportunity to partner with a PhD student (Wiseman) working under co-PD Dr. Dave Gent who was studying hop powdery mildew susceptibility genes of the MLO family. Considering that the function of these genes was so similar to that of our original R6 target gene and that characterization of MLO using gene editing was much more feasible, we chose to pivot our focus toward using gene editing technology to facilitate characterization of MLO genes. Transforming hop has been more challenging than we initially anticipated. Though we were able to achieve high rates of plant regeneration without transformation, T-DNA transfer rates have been relatively low and marker selection has been problematic. In spite of this, we have had some success in production of transgenic shoots and are working to produce a robust protocol with increased transformation frequency. We have also produced gene editing constructs for mutating our new MLO gene targets and have been working to attain plants transgenic for these CRISPR constructs. Furthermore, this project has led to efforts to increase T-DNA transfer rates from Agrobacterium in difficult-to-transform plant species such as hop. Key Personnel Willig has been awarded a postdoctoral fellowship that is focused toward this end. Also, as noted in prior progress reports, based on contemporary literature reports as well as our own experimental outcomes, we no longer believe that the use of "relaxed selection" is a viable method for achieving transgene-free gene editing in hop. Thus, we are instead focusing on transgene excision systems as an alternative mechanism for attaining transgene-free mutations. We have carried out some experiments in hop using these systems, which have been successful in our work in poplar transformation. We will employ and further develop these systems along with gene editing for MLO in hop as we begin carrying out the work funded by TASC. What opportunities for training and professional development has the project provided?The postdoctoral scholar assigned to this project (Willig), has continued to receive mentorship from the PI (Strauss), and has had opportunities for advancement of skills including experimental design, data analysis, written and oral presentation, grantsmanship, and mentorship of undergraduate student researchers, directly resulting from this project. Both Strauss and Willig have had chances to network with members of the hop growing and brewing industries, leading to additional opportunities for potential funding. Undergraduate students working on this project have also received direct training in tissue culture techniques, molecular biology theory, and other scientific competencies. Willig has greatly expanded his network and built trust within the hop growing and brewing communities. His development in grant proposal writing has led to him being awarded a 2-year postdoctoral fellowship through NIFA to develop Agrobacterium strains as better transformation tools. His grantsmanship has additionally led to our research group being awarded a 5-year grant from the Foreign Agricultural Service-Technical Assistance for Specialty Crops (TASC) program to continue developing gene editing technology in hop while partnered with the hop industry group Hop Research Council. How have the results been disseminated to communities of interest?Throughout the reporting period, dissemination of our work has been primarily targeted toward hop and brewing industry stakeholders through poster and PowerPoint presentations at industry meetings attended by the PI and postdoctoral scholar as well as podcast appearances by both (see Publications for details). We have also discussed our work and potential research outcomes in greater detail with key members of the hop industry who have an interest in seeing our research continued. All this has led to directly to a shift in perception of biotechnology among these stakeholder groups as well as to the opportunity to partner with the Hop Research Council in attaining a ~$2,000,000 grant funded through TASC, which will essentially allow continuation and expansion of the goals of this closing project with direct buy-in and participation of stakeholders. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? For the first objective under goal 1, we built 7 constructs for targeting each of the candidate R genes within two months of the project start. Since we altered our project aim (see below) to target other hop gene sources of resistance to powdery mildew, we generated an additional 6 constructs targeting candidate MLO genes with expression elements we had previously determined to best enable recovery of transgenic plants. The second objective has been completed and we have determined the hop genotypes that are most amenable to Agrobacterium transformation. Out of 8 cultivars tested, 2 ('Cascade' and 'Fuggle') were best suited to in vitro culture and transformation. Additionally, our experimental work showed that GFP is generally a poor marker gene in hop due to high levels of tissue autofluorescence. Furthermore, we determined that a spectinomycin selectable marker allowed the most efficient recovery of transgenic shoots. Thus, we switched the final iteration of our gene editing constructs to incorporate a red fluorescent protein marker (DsRed2) and a spectinomycin selectable marker. For objective three, we conducted dozens of experiments testing various tissue culture media amendments, culture incubation conditions, and inoculation methods. This work led us to incremental improvements in our transformation protocol, though the overall rate of recovery for transgenic plants is still fairly low. We did not achieve objectives four and five during this project reporting period, as no edited regenerant shoots have thus far been produced. However, we will continue to make progress on these objectives in the near future as we have secured an additional source of funding to achieve these same goals. Under goal 2, since we pivoted from the relaxed selection route for transgene-free editing to a transgene excision-based approach, we did not follow the objectives under it as stated. Instead, we evaluated methods in hop in which excision could be hypothetically applied following transgene insertion. Two of these methods in particular, altruistic regeneration induced by morphogenic genes and hairy root induction were quite promising. However, since CRISPR events with an inserted transgene were never generated, we have not yet demonstrated transgene excision in hop. However, we will continue to develop these tools in future work as well under subsequent grant funding.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Strauss, S.H., & Willig, C.J. Potential for CRISPR/gene-editing in hop breeding [conference presentation]. Master Brewers National Meeting. 2023 Oct 6-8. Seattle, WA. https://biotechlab.forestry.oregonstate.edu/sites/default/files/CRISPR_options_BrewersMtg_Oct23.pdf
• Type: Other Status: Published Year Published: 2023 Citation: Willig, C.J. Using Gene Editing to Advance Hop Research - Episode 132 [podcast appearance]. The Br� Lab Podcast. 2023 Nov 7. https://brulosophy.com/podcasts/the-bru-lab/
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Burns, L., Browning, M., Fox, G.P., Willig, C.J., Pitra, N. Exploring the Boundaries of Innovation: Biotechnology and Brewing Ingredients [Conference presentation and panel]. Craft Brewers Conference. 2024 Apr 22-24. Las Vegas, NV. https://www.craftbrewersconference.com/2024-presentations?utm_source=sfmc&utm_medium=email&utm_campaign=BA
• Type: Other Status: Submitted Year Published: 2024 Citation: Strauss, S.H., Stevens, J.F., & Willig, C.J. PlantTransform: Metabolic Engineering of Hop for Xanthohumol Enrichment [grant proposal, resubmission]. USDA-NIFA-AFRI Foundational and Applied Science. August 15, 2024. NIFA proposal no.: 2024-08865)
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Burns, L., Fox, G.P., Morrissy, C., & Willig, C.J. Genes and Genomics [Pre-conference workshop]. World Brewing Congress. 2024 Aug 17. Minneapolis, MN.
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Willig, C.J., Wiseman, M.S., Gent, D.H., & Strauss, S.H. Development of Gene Editing Methods to Retain Access to Foreign Markets for American Hops [conference presentation]. Hop Research Council Summer Meeting. 2024 Aug 6-7; Corvallis, OR. https://biotechlab.forestry.oregonstate.edu/sites/default/files/WilligHRC2024%20presentation.pdf
• Type: Other Status: Published Year Published: 2024 Citation: Willig, C.J. Hop Gene Editing at OSU - Episode 315 [podcast appearance]. Master Brewers Podcast. 2024 Aug 26. https://www.masterbrewerspodcast.com/315

Progress 07/01/22 to 06/30/23

Outputs
Target Audience:The target audiences we have primarily reached during the second year of this grant includes stakeholders in the hop growing and brewing industries through poster and speaking presentations at multiple regional and national conferences as well as through one-on-one meetings with industry representatives. Lead PI Strauss has attended the 2022 Brewing Summit in Providence, RI (hosted jointly by the Master Brewers Association of the Americas and the American Society of Brewing Chemists). There, he was invited to give an oral presentation on the implications of biotechnology in hop as it relates to brewing, as well as to participate in a panel discussion with other researchers involved in biotechnology with respect to other brewing ingredients. We also presented a poster at this conference, which communicated our project's progress and goals, and potential utility in non-technical language for the purpose of disseminating our research to the largely brewing industry audience in attendance. Dr. Strauss will participate in a similar session at the 2023 Master Brewers Conference in Seattle, WA, October 6-8. He was also invited to participate in an episode of the Master Brewers Podcast to discuss our research and the larger scope of hop biotechnology in brewing. Key personnel (Willig) has presented updates on the project research at the Washington Hop Commission annual meeting and the Hop Research Council winter meeting for audiences of hop growers, merchants, and brewing representatives. Willig was invited back to the Hop Research Council summer meeting to present and participate in a facilitated research discussion panel on potential applications of gene editing in hop. This project has led to training of three undergraduate researchers including both hands-on laboratory training and informal instruction in plant transformation and molecular biology theory. One of these student researchers has been specifically designated to work on this project (averaging ~15 hrs/wk) for the full reporting period Changes/Problems:As discussed in the previous annual report, we encountered difficulties which limited the feasibility of characterizing the R6 candidate genes we originally intended to target for gene editing. The hop varieties that were amenable to regeneration and transformation in our experiments were not ones that display the R6 dominant phenotype. Furthermore, hop genomic sequences assembled since the start of the project showed the R6 candidate locus to be highly variable and to display copy number variation across genotypes, making the sequence(s) that should be targeted uncertain without additional targeted sequencing. In light of these challenges, we chose to focus our efforts on using gene editing technology to facilitate characterization of other genes functioning in hop powdery mildew susceptibility (of the MLO family) in collaboration with co-PI Dr. David Gent and his PhD student Michele Wiseman. Transforming hop has been more challenging than we initially anticipated. Though we were able to achieve high rates of plant regeneration without transformation, T-DNA expression rates have been relatively low and marker selection has been problematic. In spite of this, we have had some success in production of transgenic shoots and are working to produce a robust protocol with increased transformation frequency. We have also produced gene editing constructs for mutating our new gene targets and experiments employing these in transformation are currently underway. Also, as noted in the previous annual report, based on contemporary literature reports as well as our own experimental outcomes, we no longer believe that the use of "relaxed selection" is a viable method for achieving transgene-free gene editing in hop. Thus, we are instead focusing on transgene excision systems as an alternative mechanism for attaining transgene-free mutations. We have carried out some initial experiments in hop using these systems, which have been successful in our work in poplar transformation. We will continue to employ these systems along with gene editing in hop during the next reporting period. What opportunities for training and professional development has the project provided?The postdoctoral scholar assigned to this project (Willig), has continued to receive mentorship from the PI (Strauss), and has had opportunities for advancement of skills including experimental design, data analysis, written and oral presentation, grantsmanship, and mentorship of undergraduate student researchers, directly resulting from this project. Both Strauss and Willig have had chances to network with members of the hop growing and brewing industries, leading to additional opportunities for potential funding. Undergraduate students working on this project have also received direct training in tissue culture techniques, molecular biology theory, and other scientific competencies. How have the results been disseminated to communities of interest?During the past reporting period, dissemination of our work has been primarily focused on hop and brewing industry stakeholders through poster and PowerPoint presentations at industry meetings attended by the PI and postdoctoral scholar (see Publications for details). We have also discussed our work and potential research outcomes in greater detail with key members of the hop industry who have an interest in seeing our research continued. This has led to potential for future work coordinating closely with the stakeholders as well as a grant proposal in coordination with the Hop Research Council. What do you plan to do during the next reporting period to accomplish the goals?Concerning goal 1, over the next reporting period, we will continue to test tissue culture parameters and new transformation techniques to attempt to increase the efficiency of transformation in hop and to ultimately produce a robust and repeatable system. In collaboration with co-PI Dr. Gent and his PhD student Michele Wiseman, we will continue gene editing work to generate target gene knockouts, estimate the efficiency of editing in hop, and then phenotype edited plants for powdery mildew susceptibility traits as we originally proposed. In service of goal 2, we will continue to test transgene excision tools in hop, induced by either a chemical or temperature-triggered signal. This work will be informed by ongoing experiments in poplar tissue to implement and improve these tools. Ultimately, we plan to build constructs that will enable genomic insertion of a transgene that will generate a targeted CRISPR/Cas9 edit, followed by induced excision of that transgene. We also aim to publish the results of our work in a peer-reviewed publication.

Impacts
What was accomplished under these goals? For the first objective under goal 1, we built 7 constructs for targeting each of the candidate R genes within two months of the project start. Since we altered our project aim (see below) to target other hop gene sources of resistance to powdery mildew, we generation 4 constructs targeting this set of candidate genes within this reporting period. The second objective has been completed and we have determined the hop genotypes that are most amenable to Agrobacterium transformation. Additionally, our experimental work showed that GFP is generally a poor marker gene in hop due to high levels of tissue autofluorescence. We continue to make progress under objectives three and four as we continue to refine our transformation system and deliver gene editing constructs in these experiments. For goal 2, progress is underway in testing methods for transgene-free gene editing, though we have also modified our approach to that (described below). We are testing systems for transgene excision in hop, which we hope to then combine with gene editing once that system has been optimized.

Publications

• Type: Other Status: Published Year Published: 2022 Citation: Willig, C.J., Wiseman, M.S., Henning, J.A., Gent, D.H., Shellhammer, T., & Strauss, S.H. Gene editing in hops: Methods, possibilities, and progress. Poster presented at: Brewing Summit 2022 (ASBC and Master Brewers joint meeting); 2022 Aug 14-16; Convention Center, Providence, RI. https://people.forestry.oregonstate.edu/steve-strauss/sites/people.forestry.oregonstate.edu.steve-strauss/files/MBAA_summit_poster2022.pdf
• Type: Other Status: Published Year Published: 2022 Citation: Strauss, S.H. & Shellhammer, T. Gene Editing for Brewers - Episode 263 [podcast appearance]. Master Brewers Podcast. 2022 Oct 17. https://www.masterbrewerspodcast.com/263
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Willig, C.J., Wiseman, M.S., & Strauss, S.H. Toward gene editing in hop: Application of a cutting-edge tool for accelerating breeding and improving traits [Conference presentation]; Washington Hop Commission annual meeting AND Hop Research Council Winter Meeting; 2023 Jan 5 AND 2023 Jan 24-27 (respectively); Yakima, WA AND Santa Rosa, CA (respectively). https://biotechlab.forestry.oregonstate.edu/sites/default/files/HRC_winter2023.pdf
• Type: Other Status: Under Review Year Published: 2023 Citation: Hop Research Council. Development of gene editing methods to retain access to foreign markets for American hops [grant proposal]. USDA-FAS Technical Assistance for Specialty Crops (TASC). May 19, 2023.
• Type: Other Status: Under Review Year Published: 2023 Citation: Strauss, S.H., & Willig, C.J. Gene editing to modify alpha acid biosynthesis in hop [grant proposal]. Hop Research Council. June 20, 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Willig, C.J., Wiseman, M.S., & Strauss, S.H. Gene editing as a powerful tool to advance hop research and agriculture [Conference presentation]; Hop Research Council Summer Meeting; 2023 Aug 1-2; Boise, ID. https://biotechlab.forestry.oregonstate.edu/sites/default/files/HRC_summer2023.pdf
• Type: Other Status: Under Review Year Published: 2023 Citation: Strauss, S.H., Stevens, J.F., & Willig, C.J. PlantTransform: Metabolic Engineering of Hop for Xanthohumol Enrichment [grant proposal]. USDA-NIFA-AFRI Foundational and Applied Science. August 16, 2023. NIFA proposal no.: 2023-08198)
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Strauss, S.H., Willig, C.J., Wiseman, M.S., Gent, D.H., Henning, J.A., & Shellhammer, T. Genetic modification methods: Basics and application to hops [Conference presentation]; Brewing Summit 2022 (ASBC and Master Brewers joint meeting); 2022 Aug 14-16; Convention Center, Providence, RI. https://people.forestry.oregonstate.edu/steve-strauss/sites/people.forestry.oregonstate.edu.steve-strauss/files/Strauss_BrewingSummit2022.pdf

Progress 07/01/21 to 06/30/22

Outputs
Target Audience:The target audience we have reached during the first year of this grant includes both the plant biotechnology research community and the brewing industry through poster and speaking presentations at two separate conferences. We presented a poster at the Society for In Vitro Biology 2022 meeting in San Diego, CA on the progress we have made so far in establishing a regeneration and transformation system in US hop varieties. Lead PI Strauss also attended the 2022 Brewing Summit in Providence, RI (hosted jointly by the Master Brewers Association of the Americas and the American Society of Brewing Chemists). There he gave an oral presentation on basic biotechnology concepts, market, social, and regulatory considerations surrounding biotech products, and how biotechnology is currently and could potentially benefit the brewing industry. He also presented a poster at this conference, which communicated our project's progress and goals, and potential utility in non-technical language for the purpose of disseminating our research to the largely brewing industry audience at this conference. This project has also led to training of three undergraduate researchers including both hands-on laboratory training and informal instruction in molecular biology theory. One of these student researchers has been specifically designated to work on this project (~20 hrs/wk) for the past 3 months of the reporting period. Changes/Problems:As stated above, we have encountered an unexpected challenge in that the hop genotypes displaying the trait we planned to genetically characterize all performed poorly compared to other genotypes in the original regeneration screen and in several follow-up experiments. Although we plan to attempt a few other experiments to attempt to overcome the low regeneration frequencies in these varieties, there is a possibility that knockout of the R6 candidate gene will turn out to be intractable in these cultivars. In that case, we have a different candidate gene in mind, which also functions in powdery mildew pathogenesis and would have the benefit of being possible to characterize in any genetic background where we can establish a gene editing system. Another change we may make to our experimental plan is in our approach to fulfilling goal 2: "Test and apply a method for obtaining CRISPR-edited hop plants that are transgene-free." A method similar to the one described in our grant proposal was published recently for another clonally propagated crop and did not show promise as a method of reliably producing transgene-free edited events. However, there are other approaches to achieving this outcome, including ongoing research in our laboratory using excision to remove CRISPR transgenes for their integration site after editing has occurred. If significant progress is made on this front by the time we are ready to proceed to goal 2, we may choose to test the efficacy of this system (still targeting the PDS gene) in hop rather than the "relaxed selection" scheme in our proposal, as this would still support the purpose behind fulfilling goal 2. What opportunities for training and professional development has the project provided?The postdoctoral scholar assigned to this project (Willig), in addition to receiving mentorship from the PI (Strauss), has been afforded opportunities for mentorship of undergraduate student researchers, oral communication skills and networking opportunities through attendance at scientific conferences and monthly project management meetings, and collaboration through with graduate student performing some work in parallel (Wiseman). Postdoctoral scholar has also gained field experience in mapping and collecting germplasm accessions. The graduate student has also been provided with training in laboratory technique, oral presentation, and grantsmanship skills as a result of this project. An undergraduate student has also received direct training in tissue culture techniques, molecular biology theory, and other scientific competencies. How have the results been disseminated to communities of interest?The results of the research performed so far on this project have been disseminated to both a relevant scientific research community (In vitro plant biology) and industry stakeholders (brewing industry professionals) through posters and oral presentations given at conferences attended by the PI and postdoctoral scholar (see Publications for details). What do you plan to do during the next reporting period to accomplish the goals?In order to proceed to the later objectives under goal 1 for characterizing the putative R6 gene through generation of CRISPR-induced knockout mutants, we need to first establish a transformation/gene editing protocol in one of the hop cultivars that display the V6 powdery mildew resistance phenotype. However, of the three cultivars tested that display this phenotype, all of them have shown poor regeneration rates in all treatments tried so far. We plan to continue testing treatments to improve baseline regeneration rates in these varieties. At the same time, we plan to improve methods for transgenic event production in our top-performing varieties and then eventually implement editing of a target gene in these cultivars using CRISPR. If we are not able to improve regeneration conditions in one of the R6 cultivars, we may choose a different target gene candidate that is also involved in the hop-powdery mildew interaction. Once we have a robust CRISPR/Cas9 editing system in any variety, we plan to pursue the objectives under goal 2 targeting the PDS gene in that genetic background.

Impacts
What was accomplished under these goals? The first objectives under goal 1: "Build multiple binary plasmid constructs carrying CRISPR/Cas9 editing reagents designed for targeting each of several candidate R genes potentially responsible for conferring a major source of resistance to hop powdery mildew (PM)" was accomplished within two months of the reporting period. The second objective: "Test multiple hop cultivars for tissue culture regeneration response as well as capacity for transient Agrobacterium-mediated transformation, measuring the latter using the expression of GFP contained in the designed plasmid constructs" is mostly complete, but still in progress. Two cultivars so far have shown a good capacity for regeneration. Progress is underway on the third objective of goal 1: "For 1-2 top-performing cultivars, optimize regeneration/transformation efficiency by varying tissue cultures parameters and transformation techniques" where we have tested and will continue to test many variations in media composition, culture conditions, and transformation techniques to improve regeneration/transformation in the best-performing cultivars we have identified ('Cascade' and 'Fuggle').

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Willig, C.J., Wiseman, M.S., Henning, J.A., Gent, D.H., & Strauss, S.H. Development of an in vitro Regeneration and Transformation System for Hop (Humulus lupulus); Poster presented at: In Vitro Biology 2022. Society for In Vitro Biology; 2022 Jun 4-7; San Diego, CA. https://sivb.org/meetings/wp-content/uploads/2022/06/Addendum-Booklet-22-updated.pdf
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Strauss, S.H., Willig, C.J., Wiseman, M.S., Gent, D.H., Henning, J.A., & Shellhammer, T. Genetic modification methods: Basics and application to hops [Conference presentation]; Brewing Summit 2022 (ASBC and Master Brewers joint meeting); 2022 Aug 14-16; Convention Center, Providence, RI. https://people.forestry.oregonstate.edu/steve-strauss/sites/people.forestry.oregonstate.edu.steve-strauss/files/Strauss_BrewingSummit2022.pdf
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Willig, C.J., Wiseman, M.S., Henning, J.A., Gent, D.H., Shellhammer, T., & Strauss, S.H. Gene editing in hops: Methods, possibilities, and progress. Poster presented at: Brewing Summit 2022 (ASBC and Master Brewers joint meeting); 2022 Aug 14-16; Convention Center, Providence, RI. https://people.forestry.oregonstate.edu/steve-strauss/sites/people.forestry.oregonstate.edu.steve-strauss/files/MBAA_summit_poster2022.pdf