Source: UNIVERSITY OF CALIFORNIA, BERKELEY submitted to NRP
METABOLIC AND CELL CYCLE CONTROL IN OBLIGATE PLANT PARASITISM
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
1016994
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2018
Project End Date
Sep 30, 2023
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF CALIFORNIA, BERKELEY
(N/A)
BERKELEY,CA 94720
Performing Department
Plant Biology, Berkeley
Non Technical Summary
Plant disease research has strong economic importance to society, as plants are the primary producers we rely on for food, textiles, air quality, and ornamental value. Climate change is expected to dramatically impact global agriculture because this industry is highly dependent on weather and pest and pathogen landscapes are already changing significantly. Therefore, to predict and maintain/enhance agricultural productivity in a changing climate, we need to understand the mechanisms underlying plant disease.Powdery mildews are widespread obligate fungal pathogens that significantly impact crop and forest product yield and quality. For example, California grapevines are standardly treated 9-11 times per season to reduce powdery mildew disease and heavily infected oaks can exhibit as high as 70-90% reduction in annual radial growth. Furthermore, fragmented forested areas and climate change favor powdery mildew disease. As obligate biotrophs, powdery mildews only grow and reproduce on living plant tissue. They draw all their nutrient resources from the plant, with an associated impact on plant yield and quality. Their genomes reflect their obligate dependence in that they have lost core metabolic pathways as well numerous genes involved in secondary metabolism. Other obligate plant biotrophs from evolutionarily distinct lineages have lost overlapping sets of metabolic pathways and genes providing evidence for selective loss and convergent evolution. We are using molecular genetic and genomics, cell biology, metabolic biochemistry, and modeling approaches to determine the plant metabolites required by the powdery mildew at each stage of its infection process and the means by which the powdery mildew manipulates the plant to achieve this goal. This includes powdery mildew manipulation of the plant host cell cycle. Assessment of conserved powdery mildew targets in grapevine will allow for direct application of our findings.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2122410101025%
2151131104025%
2064020103025%
1232499110225%
Goals / Objectives
Powdery mildews are widespread obligate fungal pathogens that significantly impact crop and forest product yield and quality (Glawe et al. 2012; Fuller et al. 2014; Bert et al., 2016). For example, California grapevines are standardly treated 9-11 times per season to reduce powdery mildew disease and heavily infected oaks can exhibit as high as 70-90% reduction in annual radial growth (Bert et al. 2016). Furthermore, fragmented forested areas and climate change favor powdery mildew disease (Bebber et al. 2014; Jousimo et al. 2014).As obligate biotrophs, powdery mildews only grow and reproduce on living plant tissue. They draw significant nutrient resources from the plant, with an associated impact on plant yield and quality. Their genomes reflect their obligate dependence in that they have lost core metabolic pathways as well numerous genes involved in secondary metabolism (Spanu et al. 2010). Other obligate plant biotrophs from evolutionarily distinct lineages have lost overlapping sets of metabolic pathways and genes providing evidence for selective loss and convergent evolution (Spanu 2012). Not only are plant metabolites critical as nutrients for these obligate biotrophs, but they can also play signaling roles and direct developmental processes. For example, we found a plant PDH-bypass derived product (likely an Acetyl-CoA derivative) controls the initiation of powdery mildew asexual reproduction.In the G. orontii-Arabidopsis interaction, we found the powdery mildew induces a variant of the cell cycle called endoreduplication resulting in a 32-fold increase in DNA content in the mesophyll cells underlying the fungal feeding site (Chandran et al. 2010; Chandran et al. 2013). Arabidopsis mutants compromised in endoreduplication in these cells support fewer asexual reproductive structures (conidiophores) per colony. Furthermore, the DNA content of the mesophyll cells underlying the fungal feeding site is highly correlated with the extent of asexual reproduction for this series of mutants indicating induced endoreduplication acts as a susceptibility determinant for this interaction (Chandran et al. 2013).Plant biotroph-induced localized endoreduplication appears to be a shared strategy for increasing localized host metabolic capacity to increase nutrient supply during metabolically demanding phases of biotroph growth and development (Wildermuth 2010; Chandran et al. 2016; Wildermuth et al. 2017). In addition to powdery mildew, diverse biotrophs including Sinorhizobia, arbuscular mycorrhizal fungi, cabbage leaf curl virus, and root nematodes induce endoreduplication (Wildermuth et al. 2017). Although increased DNA content has long been associated with enhanced metabolic capacity (Nagl 1976), the specific pathways and mechanisms are not well defined.Transcriptome analysis identified metabolic processes that were preferentially enhanced across systems with induced endoreduplication in a developmental or biotroph/plant interaction context (Wildermuth, 2010). Our further analysis including molecular genetic, plant pathology, and lipid analyses found the plant PDH bypass to be endoreduplication-associated and to be critical to powdery mildew asexual reproduction. Not only do PDH bypass product(s) serve to provide lipid precursors for lipid body formation in developing spores, but also act to control the number of asexual reproductive structure formed therefore acting as a developmental signal linking nutrient (lipid) availability with reproductive output. Lipids act as energy dense molecules and their acquisition from hosts has now been shown to be critical to diverse plant and animal biotrophs/intracellular pathogens including arbuscular mycorrhizal fungi (e.g. Kewer et al. 2017) and Mycobacterium tuberculosis (reviewed in Toledo and Benach, 2015).By combining molecular genetic/genomic, plant pathology, modeling, metabolic and analytical chemistry, and modeling approaches we seek to:1. Uncover specific plant metabolic pathways, enzymes, and compounds required for powdery mildew colonization, growth and reproduction in an infection-phase specific manner,2. Uncover mechanisms by which the powdery mildew locally redirects the plant host cell cycle and metabolism to promote its colonization, growth and reproduction,3. Identify shared strategies, process hubs and targets in common to obligate plant biotrophs, and4. Translate our findings in Arabidopsis to grapevine, for which powdery mildew disease is particularly problematic, and with future funding to forest species particularly susceptible to powdery mildews.Secondary Project: Salcylic acid and plant defense vs. growthSalicylic acid (SA) is the key phytohormone controlling plant defense to (hemi)biotrophic pathogens and the induction of plant immunity. However, the full biosynthetic pathway has not yet been resolved. In addition, modification of SA controls is activity and function. Induction of SA is tightly regulated as misregulated enhancement of SA inhibits plant growth. My lab plans to build on our previous body of work in this area (e.g. Wildermuth et al. 2001; Strawn et al. 2007; Nobuta et al. 2007; Okrent et al. 2009; Dempsey et al. 2011; Chandran et al. 2014; Mackelprang et al. 2017; Seguel et al. 2018) and usemolecular genetic/genomic, plant immunity/defense, cell biology, biochemistry, and modeling approaches to:1. Fully define the SA metabolic pathway via isochorismate2. Uncover mechanisms by which SA modification controls specific pathogen and immunity outcomes3. Elucidate key players and regulatory strategies involved in SA/immunity cell cycle control and growth suppression
Project Methods
Metabolic and cell cycle control in obligate plant parasitismAims 1 &2: I have established acollaboration with Sue Rhee (Carnegie Institute for Science) to utilize our powdery mildew and associated host genome to create an integrated genome-reconstructed metabolic network. This has been created using the draft G. orontii MGH v.1 genome with the host Arabidopsis thaliana. We now have an optimized genome that includes HiC data. The integrated metabolic network will be revised with the optimized G. orontii MGH1 v.3 genome, and together the Rhee and Wildermuth group will do rigorous QC and manual correction. We have performed time series RNA_Seq of the powdery mildew infection of Arabidopsis and are awaiting the data for analysis. Infection-phase specific data (e.g. germination, feeding structure formation, asexual reproduction) will then be overlayed on the integrated metabolic network for both the powdery mildew and Arabidopsis to predict infection phase specific use of Arabidopsis metabolic pathways and products by the fungus. We have already identified a subset of these by hand which we are testing experimentally using Arabidopsis mutants and by silencing powdery mildew genes using spray-induced gene silencing (SIGS). With the Rhee group, we will also perform constraint-based modeling to identify metabolic pathways/products that drive outcomes under particular conditions.With collaborator Trent Northen, at JGI, we have shown that the powdery mildew uses the plant PDH bypass to provide lipid precursors which are incorporated into lipid storage compounds (triacylglycerols) in developing spores. The Northern lab has now developed protocols and tools for exometabolite analysis on whole Arabidopsis plants, which we can use to identify plant metabolites taken up by the fungus at specific infection phases. Altering the predicted associated metabolic pathway and transport genes in Arabidopsis and/or powdery mildew will be done using molecular genetic approaches (e.g. mutants, CRISPR-Cas9, SIGS) to assess impact on powdery mildew colonization, growth, and reproduction and on specific metabolite/nutrient acquisition.To manipulate the plant cell cycle and metabolism, effectors and small interfering RNAs are likely candidates. We have identified G. orontii effectors that target Arabidopsis cell cycle proteins and transcription factors that regulate nutrient reservoir filling and lipid biosynthesis and are testing their function in planta. We will also identify additional effectors and siRNAs of interest through phase-specific transcriptome and small RNA profiling.Aim 3: We will explore whether our findings with the powdery mildew-Arabidopsis interaction translates to other obligate-biotroph interactions using comparative genomics and in vitro approaches (e.g. effector - target interactions) and/or through collaborations with colleagues working on those systems.Aim 4: We will optimize SIGS of the grape powdery mildew on grapevine first with an established conserved essential powdery mildew gene. We will then target conserved powdery mildew genes needed for nutrient acquisition/utilization and use our developed high throughput powdery mildew disease assessment assays to evaluate their contribution to powdery mildew colonization, growth, and/or reproduction.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Target audience for fundamental scientific discoveries is primarily other scientific researchers in the fields of plant biology, plant pathology, plant-microbe interactions, host-microbe interactions, plant biochemistry and metabolism, plant defense, plant hormone biology, plant development and growth, mycology, bacteriology, cancer biology, cell cycle and cell fate. Efforts to reach these audiences include presentations at (inter)national meetings, research institutes, and universities. Target audience for translational efforts include agricultural biotech and breeding companies, grapevine growers, pest control advisors, certified crop advisors, vineyard managers and consultants, and agricultural researchers in academic and government labs (e.g. USDA and DOE). Efforts to reach these audiences include presentation and participation in the Unified Grape Symposium, Agbiotech networking events at UC Berkeley and UC Davis, and participation in the National Science Foundation (NSF) innovation (i)-Corps program, a market/customer discovery program in which we interviewed 100 persons in the wine industry ecosystem including vineyard managers and consultants, vineyard owners, pest control advisors, certified crop advisors, agricultural extension specialists, fungicide distributors, regulators, organic and sustainable wine associations to understand user needs and desires for reducing powdery mildew infection of grapevine. This process allowed us to further refine our target market and verify that we are developing a product (topical RNAi to control powdery mildew of grapevine) that is needed; it also allowed us to define the minimal viable product requirements of the end-user and additional desired features. Target audience for formal teaching include undergraduates at UC Berkeley taking MCB102 Biochemistry (3 units, 430 students) and PMB101L (3 units, 18 students). I teach the MCB102 Metabolism section of MCB102 and incorporate both human and plant examples as part of my 13 lectures. For PMB101L, the capstone discovery-based laboratory course for senior plant biology majors, the students' research focused on elucidating components of the powdery mildew-plant interaction. More informal teaching includes training of three graduate students and three undergraduates. Students receive guidance on experimentation and scientific communication in oral and written formats. Target audience for outreach activities includes a program "Be A Scientist" that I founded and developed in collaboration with the non-profit Community Resources for Science for 7th graders at Berkeley Public Middle Schools. Berkeley Middle School students are very diverse with 45% receiving free/subsidizing lunch, ~22% African-American, and ~25% Latino/Latina. The Be A Scientist Program is a six-week in class program at each school in which every 7th grader is partnered with a UC Berkeley STEM graduate student mentor to design an experimental question of their choosing, test it, analyze the data, and present their findings. The program expanded to all three middle schools this past year, reaching >700 7th graders with >150 UC Berkeley scientist mentors. The Be A Scientist program teaches the skills of being a scientist, and cross-cutting next generation science standard concepts. The program has been a huge success and is funded by the Berkeley Unified School District (BSEP funds), the non-profit Berkeley Public Schools Fund, and a Chancellors' Community Project Grant. Be A Scientist received the 2020 Chancellor's Award for Campus-Community Partnership. Changes/Problems:Due to COVID-19, experimental lab time was partially limited. We used this time to develop our multi-dimensional target prediction pipeline. Much more time was also spent teaching as all coursework had to be redesigned for remote learning. What opportunities for training and professional development has the project provided?Training and Development of Undergraduates: Jenny Lee, Silverdew Shi (Honor's thesis), and Uzair Nazir.Students received supplements through the UC Berkeley Undergraduate Research Apprenticeship Program,Student-Initiated Sponsored Projects Undergraduate Research Program, and NSF Research Experience for Undergraduates program, and takeHaas business school classes in entrepreneurship. Training and Development of Graduate Students: Amanda McRae, Johan Janeisch, and Hang Xue. Amanda completed her PhD in August 2020. Their fields of training included plant biology, fungal biology, phytopathology, plant molecular biology, genetics, biochemistry, and metabolomics. Amanda was further trained in entrepreneurship and was co-Entrepreneurial Lead in our national iCorps program participation. Training and Development of Postdoctoral Researchers: Dr. Jyoti Taneja, now Assistant Project Scientist, has trained in technology development and transfer, plant pathology, leadership, and entrepreneurship. She was the Entrepreneurial Lead for the national NSF iCorps program, she has attended diverse networking events, and I have been training her in grant writing and mentorship. How have the results been disseminated to communities of interest?Dissemination of our scientific results occurred through remote UC Berkeley events, interviews/discussions with 100 persons in the wine industry ecosystem through NSF iCorps program, the Unified Grape Symposium, and other scientific, wine industry related, and entrepreneurship remote events. What do you plan to do during the next reporting period to accomplish the goals?We are patenting our IP and developing a minimal viable product - topical RNAi powdery mildew fungicide for grapevine. We are planning to multiplex our targets, perform field testing at two sites, and start partnering to test delivery systems. We will publish four papers this coming year, as we have been waiting for our patent to be filed.

Impacts
What was accomplished under these goals? IMPACT For California grapevines, $6.2B in 2018,powdery mildew is the dominant disease. Greater than75% of pesticide cost is associated with powdery mildew management, representing 4-8% of total production costs. Asresistance has emerged against current fungicides, new technologies are needed. dsRNA is natural, degrades in the environment, and is designed to be specific to the gene in the targeted organism. With its reduced health and environmental risks, topical RNAi is a very promising approach, and my lab's ongoing fundamental research on the powdery mildew-plant system has allowed us to a) obtain the required genomic information, b) develop spray-induced gene silencing (SIGS) methodology for powdery mildew, c) be highly successful in prioritizing targets for screening, now with a 94% success rate, and d) evaluate the market and define end-user needs. Efforts to translate this effort for commercialization are well underway. SPECIFIC RESULTS Powdery mildew- plant host interaction By combining molecular genetic/genomic, plant pathology, modeling, metabolic and analytical chemistry, and modeling approaches we seek to: 1. Uncover specific plant metabolic pathways, enzymes, and compounds required for powdery mildew colonization, growth and reproduction in an infection-phase specific manner, Graduate students Amanda McRae and Johan Jaenisch have discovered more than 12 additional novel plant genes that when knocked out result in altered powdery mildew reproduction. These genes include metabolic enzymes required to make nutrient precursors required by the fungus, transcription factors that mediate expression of these enzymes, and transporters. In addition, additional plant cell fate regulators that impact powdery mildew-induced endoreduplication have been identified. These all represent new targets that could be used for genome-informed grapevine or other crop breeding to reduce powdery mildew disease or for markerless CRISPR-Cas editing. These targets are all associated with decreased powdery mildew reproduction. 2. Uncover mechanisms by which the powdery mildew locally redirects the plant host cell cycle and metabolism to promote its colonization, growth and reproduction, Amanda McRae and Johan Jaenisch have identified novel plant host cell cycle/fate regulators that mediate the powdery mildew - plant interaction, eg resulting in reduced powdery mildew reproduction. Amanda has shown that specific powdery mildew effectors interact with these plant host cell cycle/fate regulators. Furthermore, silencing these powdery mildew effector genes also reduces powdery mildew reproduction. Amanda has further characterizing this effector-plant target node of response and identified a conserved effector with specialized function in powdery mildews. By expressing tagged versions of this powdery mildew effector in plants, she has co-immunoprecipitated associated interacting powdery mildew and plant host proteins. These include cell cycle-related proteins and others that coordinate cell fate, defense, and energy. These powdery mildew and host genes can be targets for limiting powdery mildew disease. Johan is further pursuing ploidy-associated changes in host plant metabolism using the powdery mildew system including lipid analysis and modeling in combination with the Northen (JGI) and Rhee (Carnegie) labs. He has identified specific changes in host plant lipid metabolism associated with powdery mildew infection when host lipids are needed to provide resources for powdery mildew spore formation. [Spores are filled with lipid bodies, used to fuel subsequent germination.] Much of what he has observed relates to seed filling and/or manipulation of plants to enhance fatty acid/oil production. Therefore, this aspect of the work is relevant to both obligate plant biotrophs, many of which acquire lipids from the plant host, to fuel their own development, and to optimizing plant oil production. 3. Identify shared strategies, process hubs and targets in common to obligate plant biotrophs, and Comparative genomics and transcriptomics of powdery mildews based on the data obtained through our DOE JGI CSP #1657 Comparative Genomics of Powdery Mildews and their Associated Hosts, allowed us to identify conserved powdery mildew proteins of interest associated with specific phases of powdery mildew infection, growth, and reproduction. Coupling these analyses with evolutionary analysis, metabolic pathway analysis and modeling, and our deep knowledge of the powdery mildew-plant interaction and its requirements, we prioritized genes for study by process category. We now have 40 conserved powdery mildew genes that when silenced via topical RNA result in significantly reduced powdery mildew growth and reproduction. Furthermore, we identified specific process categories that are particularly productive and reflect the metabolic dependencies of the powdery mildew on the plant host. These targets should translateto other plant obligate biotrophs which have similar dependencies on the plant. ?Dr. Taneja and graduate student Amanda McRae were involved in the above efforts with bioinformatic support from first year (rotating) graduate student Kyungyong Seong. 4. Translate our findings in Arabidopsis to grapevine, for which powdery mildew disease is particularly problematic, and with future funding to forest species particularly susceptible to powdery mildews. We developed spray-induced gene silencing of powdery mildew genes using the Arabidopsis-powdery mildew system and showed that conserved powdery mildew gene targets that when silenced result in reduced powdery mildew reproduction of Arabidopsis also result in reduced powdery mildew in the grapevine system. Forty novel powdery mildew targets have now been identified - and eleven verified in grapevine. We also performed our first field test in grapevine, in parallel with Dr. Akif Eskalen's fungicide trial at the Trinchero Vineyards Sacramento Delta site. We had 10 grapevine per treatment type, control and untreated, and five different treatments. Unfortunately, it was a rare year at this site, in that the powdery mildew disease was so low we could not accurately assess differences with our treatment. However, we did learn how to do a grapevine field trial, that our RNAi fungicide is compatible with commercial sprayers, that it does not impact canopy size or berry development, and that it is not phytotoxic. Dr. Taneja led our field trial efforts and three undergraduates have been associated with this project. Through our NSF Partners for Innovation -Technology Transfer grant, we also participated in the national NSF iCorps program - an intensive 8-week market/customer discovery program with Dr. Taneja as Entrepreneurial Lead (EL), Amanda McRae as co-EL, Dr. Wildermuth as Technical Lead, and Kevin Hammill (COO, Maronne Bio Innovations) as Industrial Mentor. As part of this process, we interviewed 100 persons in the wine industry ecosystem including vineyard managers and consultants, vineyard owners, pest control advisors, certified crop advisors, agricultural extension specialists, fungicide distributors, regulators, organic and sustainable wine associations to understand user needs and desires for reducing powdery mildew infection of grapevine. This process allowed us to further refine our target market and verify that we are developing a product (topical RNAi to control powdery mildew of grapevine) that is needed. It also allowed us to define the minimal viable product requirements of the end-user and additional desired features. We further talked to RNA scale up and formulation startups and companies as well as the few companies developing RNAi products for agricultural use. We had clear interest from them in our target portfolio. Secondary Project: Salcylic acid and plant defense vs. growth: Nothing to report

Publications


    Progress 10/01/18 to 09/30/19

    Outputs
    Target Audience:Target audience for fundamental scientific discoveries is primarily other scientific researchers in the fields of plant biology, plant pathology, plant-microbe interactions, host-microbe interactions, plant biochemistry and metabolism, plant defense, plant hormone biology, plant development and growth, mycology, bacteriology, cancer biology, cell cycle and cell fate. Efforts to reach these audiences include presentations at (inter)national meetings, research institutes, and universities. Target audience for translational efforts include agricultural biotech and breeding companies, grapevine growers, vineyard managers and consultants, and agricultural researchers in academic and government labs (e.g. USDA and DOE). Efforts to reach these audiences include presentation and participation in the UC Davis Continuing and Professional Education course "Current Wine and Winegrape Research", Agbiotech networking events at UC Davis, and conversations with vineyard managers and consultants, and agricultural extension specialists to understand user needs and desires for reducing powdery mildew infection of grapevine. Target audience for formal teaching include undergraduates at UC Berkeley taking MCB102 Biochemistry (3 units, 450 students) and PMB101L (3 units, 16 students). I teach the MCB102 Metabolism section of MCB102 and incorporate both human and plant examples as part of my 13 lectures. For PMB101L, the capstone discovery-based laboratory course for senior plant biology majors, the students' research focused on elucidating components of the powdery mildew-plant interaction. More informal teaching includes training of three graduate students and three undergraduates. Students receive guidance on experimentation and scientific communication in oral and written formats. Target audience for outreach activities includes a program "Be A Scientist" that I founded and developed in collaboration with the non-profit Community Resources for Science for 7th graders at Berkeley Public Middle Schools. Berkeley Middle School students are very diverse with 45% receiving free/subsidizing lunch, ~22% African-American, and ~25% Latino/Latina. The Be A Scientist Program is a six-week in class program at each school in which every 7th grader is partnered with a UC Berkeley STEM graduate student mentor to design an experimental question of their choosing, test it, analyze the data, and present their findings. The program expanded to all three middle schools this past year, reaching >700 7th graders with >150 UC Berkeley scientist mentors. The Be A Scientist program teaches the skills of being a scientist, and cross-cutting next generation science standard concepts. The program has been a huge success and is currently funded by the Berkeley Unified School District (BSEP funds), the non-profit Berkeley Public Schools Fund, and a Chancellors' Community Project Grant. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training and Development of Undergraduates: Three undergraduates were trained on this project. Students received supplements through the UC Berkeley Undergraduate Research Apprenticeship Program (URAP) and Student-Initiated Sponsored Projects Undergraduate Research Program (SPUR). Two undergraduates, Keshav Kumar and Elizabeth Applegate, did their honor's thesis research in the lab, presented posters on their research and graduated in May 2019. Keshav received the UC ANR Howard Walton Clark Prize. This prize is given annually to a senior student in the College of Agriculture whose high scholastic achievement, talent for independent research, and other characteristics, with particular reference to either plant breeding or soil building, seem to show the greatest promise. Training and Development of Graduate Students: Two senior graduate students and one rotating first year graduate student were directly trained and mentored. Graduate students Amanda McRae and Johan Jaenish both attended and presented their research at the International Congress on Molecular Plant-Microbe Interactions in Glasgow, Scotland (July, 2019) and the associated Powdery Mildew Symposium. In addition, they presented their research at the Departmental student/postdoc seminar series and other regional events. Furthermore, Amanda McRae organized the UC Berkeley Plant Genome Editing Symposium (May 2019) sponsored by Corteva and presented her research at this event. Their fields of training included plant biology, fungal biology, phytopathology, plant molecular biology, genetics, biochemistry,and metabolomics. Amanda was further trained in entrepreneurship and took the 4-unit UC Berkeley Challenge Lab course offered through the UC Berkeley Sutardja Center for Entrepreneurship & Technology that leads you through the venture creation project from idea to pitching to panels of investors and expert faculty. Training and Development of Postdoctoral Researchers: Dr. Jyoti Taneja, developed the grapevine powdery mildew system in the lab and has been both i) moving forward our technology and identification of novel targets for silencing powdery mildew genes to reduce powdery mildew of grapevine and ii) developing our technology transfer and market approach. She has been participating in entrepreunurial events through the UC Berkeley Bakar Innovation Fellow program, Agbiotech events at UC Davis, and networking events and meetings with grapevine stakeholders. I have also actively mentored her in leadership and supervising skills, as she oversaw two of the undergraduate students and I am grooming her as lead on the translational efforts in my laboratory. How have the results been disseminated to communities of interest?Dissemination of our scientific results occurred through UC Berkeley events (retreats and seminars) and attendance and presentation at scientific conferences and seminars/visits to other research institutes. In addition, I presented our research on spray-induced gene silencing to reduce powdery mildew disease at the UC Davis Extension Course "Current Wine and Grape Research". We also discussed our research and met with stakeholders at the UC Davis Extension Course, Agbiotech meetup events in Davis, and UC Berkeley Bakar program hosted entrepreneurial events. What do you plan to do during the next reporting period to accomplish the goals?I received an NSF Partners for Innovation Technology Transfer grant to develop a knowledge of the market for our potential product to reduce powdery mildew disease of agricultural crops and a technology transfer/commercialization path. As part of this program, we will identify an industrial mentor, build our team, and participate in the NSF national iCorps program.

    Impacts
    What was accomplished under these goals? IMPACT My laboratory developed a new technology, spray-induced gene silencing (SIGS) against powdery mildew genes to reduce powdery mildew disease. To our knowledge, we were the first to get this system working with powdery mildews which are obligate biotrophic pathogens. We developed and optimized a pipeline for target selection and assessment using an Arabidopsis-powdery mildew system and a grapevine powdery mildew system. We identified 12 novel targets that statistically and reproducibly reduce powdery mildew disease of Arabidopsis, 6 of these have also been tested in grapevine with a similar reduction in powdery mildew disease. We also received an NSF Partners for Innovation Technology Transfer award to explore the market for our product, participate in NSF iCorps, and develop entrepreneurial skills. Furthermore, we secured additional funding from the American Vineyard Foundation and we have networked with grapevine growers and breeders, vineyard owners, managers, and consultants, and agricultural biotech companies. Through presentation at the UC Davis Extension Course in Current Wine and Grape Research we informed stakeholders of our findings and learned about their specific needs (e.g. in terms of application, etc). For California grapevines, $6.2B in 2018, powdery mildew is the dominant disease. Greater than 75% of pesticide cost is associated with powdery mildew management, representing 4-8% of total production costs. As resistance has emerged against current fungicides, new technologies are needed. dsRNA is natural, degrades in the environment, and is designed to be specific to the gene in the targeted organism. With its reduced health and environmental risks, it is a very promising approach, and my lab's ongoing fundamental research on the powdery mildew-plant system has allowed us to be highly successful in prioritizing targets for screening, with a 60% success rate in SIGS against a powdery mildew gene resulting in signficantly reduced spore production. SPECIFIC RESULTS Powdery mildew- plant host interaction By combining molecular genetic/genomic, plant pathology, modeling, metabolic and analytical chemistry, and modeling approaches we seek to: 1. Uncover specific plant metabolic pathways, enzymes, and compounds required for powdery mildew colonization, growth and reproduction in an infection-phase specific manner, Graduate students Amanda McRae and Johan Jaenisch have discovered more than 10 novel plant genes that when knocked out result in altered powdery mildew reproduction. These genes include metabolic enzymes required to make nutrient precursors required by the fungus, transcription factors that mediate expression of these enzymes, and transporters. In addition, additional plant cell fate regulators that impact powdery mildew-induced endoreduplication have been identified. These all represent new targets that could be used for genome-informed grapevine or other crop breeding to reduce powdery mildew disease or for markerless CRISPR-Cas editing. These targets are all associated with decreased powdery mildew reproduction. 2. Uncover mechanisms by which the powdery mildew locally redirects the plant host cell cycle and metabolism to promote its colonization, growth and reproduction, Graduate students Amanda McRae and Johan Jaenisch have identified novel plant host cell cycle/fate regulators that mediate the powdery mildew - plant interaction, eg resulting in reduced powdery mildew reproduction. Amanda has shown that specific powdery mildew effectors interact with these plant host cell cycle/fate regulators. Furthermore, silencing these powdery mildew effector genes also reduces powdery mildew reproduction. Amanda is further characterizing this effector-plant target node of response. These powdery mildew effectors and host genes both can be agricultural targets for limiting powdery mildew disease. Johan is further pursuing ploidy-associated changes in host plant metabolism using the powdery mildew system including lipid analysis and modeling in combination with the Northen (JGI) and Rhee (Carnegie) labs. Spray-induced silencing of powdery mildew genes has allows us to systematically identify powdery mildew genes important to each phase of its infection and reproduction. Amanda and Dr. Jyoti Taneja in the lab have identified 12 novel powdery mildew genes important to powdery mildew reproduction. 3. Identify shared strategies, process hubs and targets in common to obligate plant biotrophs, and A number of the plant host regulators identified above are reported by Y2H to be targeted by effectors of other plant biotrophs. Future work will explore these commonalities. 4. Translate our findings in Arabidopsis to grapevine, for which powdery mildew disease is particularly problematic, and with future funding to forest species particularly susceptible to powdery mildews. We developed spray-induced gene silencing of powdery mildew genes using the Arabidopsis-powdery mildew system and showed that conserved powdery mildew gene targets that when silenced result in reduced powdery mildew reproduction of Arabidopsis also result in reduced powdery mildew in the grapevine system. Twelve novel powdery mildew targets have been identified - and six verified in grapevine. As discussed in impact, we are continuing this work and exploring the commercial feasibility of these RNA fungicides. Amanda has led the SIGS work using the Arabidopsis-powdery mildew systemand Dr. Taneja has led development of testing in the grapevine-powdery mildew system. Both Amanda and Dr. Taneja have also been involved in entrepreneurial efforts. Secondary Project: Salcylic acid and plant defense vs. growth Nothing to report. Additional experimental work was not performed this year due to lack of funding and associated personnel.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2019 Citation: Wildermuth MC. Plants fight fungi using kiwellin proteins. Nature. 2019 Jan;565(7741):575-577. doi: 10.1038/d41586-019-00092-2.