Deciphering pathogenicity and development in obligate downy mildew pathogen using small RNA approach.
Lead Research Organisation:
University of Worcester
Department Name: Inst of Science and the Environment
Abstract
The oomycetes comprise several hundred microbial species including unique groups of biotrophic, necrotrophic and hemibiotrophic plant pathogens. They have superficial similarity to filamentous fungi but are distinct from them in several areas: the cell walls of oomycetes have been reported to be primarily B-1-3 glucans and cellulose with little or no chitin, oomycetes' hyphae are coenocytic (multinucleate with no division by septa) and their vegetative nuclei are in a diploid state. The diseases caused by oomycete plant pathogens include seedling blights, damping-off, root rots, foliar blights and downy mildews. Collectively, oomycetes estimated to cause 10's of billions in losses annually, due to their high evolutionary potential that enables host jumps, resistance to fungicides, and suppression or evasion of host resistance genes. Some of the most economically important oomycete pathogens are Phytophthora infestans (tomato and potato late blight), P. ramorum (sudden oak death), P. capsici (stem and fruit rot of cucumber and pepper), P. cinnamomi (dieback in avocado, pineapple), Plasmopora viticola (grapevine downy mildew), P. halstedii (sunflower downy mildew), Pythium ultimum (damping off and root rot), Bremia lactuca (lettuce downy mildew), and Albugo candida (white blister rust of crucifers).
The biotrophic oomycete Hyaloperonospora arabidopsidis has co-evolved as a downy mildew pathogen in wild populations of Arabidopsis thaliana and has been used for more than 30 years as an experimental model for investigating the molecular basis of the gene-for-gene theory and other aspects of plant-oomycete interactions.
Obligate oomycetes are not amenable to genetic transformation, thus hindering genetic analysis. Several groups including ours have relied on alternative approaches to assay effector function in planta including: a) co-bombardment assays into plant cells using the GUS gene to indicate avirulence activity, b) delivering effectors using bacteria secretion system, and c) creation of stably transformed plants expressing effector genes under control of plant promoters. However, all of these methods stripped the effector gene away from the pathogen where the expression level of a gene may not be comparable to that in the native background. Moreover, single-gene assays do not accurately capture gene function in the native milieu. Finally, these approaches are only applicable to secreted effector proteins that operate inside host cells. Our approach breaks the current barriers and employs reverse genetics in obligate oomycetes by applying sRNA directly to spores to trigger gene silencing.
This innovative approach described in this project focuses on the to use of a small RNA (sRNA) approach to increase our understanding of plant - biotrophic oomycete microbe interactions. We aim to use high-throughput genetic screen to identify and study genes specifically involved in spore germination, infection, mycelial development, sporulation, nutrient uptake, and host immune suppression. We will investigate the properties of sRNA-mediated silencing, optimize, and test in other oomycetes. We will Generate gene-specific sRNAs for highly regulated genes in spores, during germination, mycelial development and sporulation. We will then apply gene specific sRNAs to identify genes showing a phenotype upon silencing. Using this technique, we will also investigate some of the well-known effector genes under native conditions. These would lead to identification and characterization of pathogen genes that could be targeted for disease control. Results obtained from this work can easily be transferred to other obligate downy mildews of grapevine, lettuce, or brassica.
The biotrophic oomycete Hyaloperonospora arabidopsidis has co-evolved as a downy mildew pathogen in wild populations of Arabidopsis thaliana and has been used for more than 30 years as an experimental model for investigating the molecular basis of the gene-for-gene theory and other aspects of plant-oomycete interactions.
Obligate oomycetes are not amenable to genetic transformation, thus hindering genetic analysis. Several groups including ours have relied on alternative approaches to assay effector function in planta including: a) co-bombardment assays into plant cells using the GUS gene to indicate avirulence activity, b) delivering effectors using bacteria secretion system, and c) creation of stably transformed plants expressing effector genes under control of plant promoters. However, all of these methods stripped the effector gene away from the pathogen where the expression level of a gene may not be comparable to that in the native background. Moreover, single-gene assays do not accurately capture gene function in the native milieu. Finally, these approaches are only applicable to secreted effector proteins that operate inside host cells. Our approach breaks the current barriers and employs reverse genetics in obligate oomycetes by applying sRNA directly to spores to trigger gene silencing.
This innovative approach described in this project focuses on the to use of a small RNA (sRNA) approach to increase our understanding of plant - biotrophic oomycete microbe interactions. We aim to use high-throughput genetic screen to identify and study genes specifically involved in spore germination, infection, mycelial development, sporulation, nutrient uptake, and host immune suppression. We will investigate the properties of sRNA-mediated silencing, optimize, and test in other oomycetes. We will Generate gene-specific sRNAs for highly regulated genes in spores, during germination, mycelial development and sporulation. We will then apply gene specific sRNAs to identify genes showing a phenotype upon silencing. Using this technique, we will also investigate some of the well-known effector genes under native conditions. These would lead to identification and characterization of pathogen genes that could be targeted for disease control. Results obtained from this work can easily be transferred to other obligate downy mildews of grapevine, lettuce, or brassica.
Technical Summary
Obligate oomycetes are not amenable to genetic transformation, thus hindering genetic analysis. Several groups including ours have relied on alternative approaches to assay effector function in planta including: a) co-bombardment assays into plant cells using the GUS gene to indicate avirulence activity, b) delivering effectors using bacteria secretion system, and c) creation of stably transformed plants expressing effector genes under control of plant promoters. However, all of these methods stripped the effector gene away from the pathogen where the expression level of a gene may not be comparable to that in the native background. Moreover, single-gene assays do not accurately capture gene function in the native milieu. Finally, these approaches are only applicable to secreted effector proteins that operate inside host cells. Movement of small RNAs from plants to pathogens has been explored using HIGS. Although we tried HIGS methods several times in Arabidopsis to study functions of ATR1, ATR13 and ATR5 for Hyaloperonospora arabidopsidis (Hpa) in our laboratory, it proved unsuccessful (Tör Group, unpublished results). However, we discovered that application of sRNA can be effectively used to elucidate gene function in Hpa. We will Investigate the properties of sRNA-mediated silencing, optimize, and test in other oomycetes including Peronospora viciae, Phytophthora parasitica and P. capsici. We will Generate gene-specific sRNAs for highly regulated genes in Hpa spores, during germination, mycelial development and sporulation. We will then apply gene specific sRNAs to identify genes showing a phenotype upon silencing. Using this technique, we will also investigate some of the well-known effector genes under native conditions. These would lead to identification and characterization of pathogen genes that could be targeted for disease control. Results obtained from this work can easily be transferred to other obligate downy mildews of grapevine, lettuce, or brassica
Publications
Bilir Ö
(2022)
Small RNA-based plant protection against diseases
in Frontiers in Plant Science
Tör M
(2023)
Recent developments in plant-downy mildew interactions
in Seminars in Cell & Developmental Biology
Description | We showed that Short-Synthesised double stranded RNA (SS-dsRNA) can be used in a high throughput manner to silence genes in an obligate oomycete downy mildew pathogen. We originally worked on the model system, Arabidopsis-downy mildew but we transferred this technology to crop plants including lettuce and pea downy mildews. Till now, we have targeted around 120 genes and showed that 40 of these genes inhibit germination and sporulation and the rest showed enhanced or decreased rate of germination and sporulation. Some of the genes also found to be involved in germ tube length and oospore (overwintering spores) formation. These findings are useful for RNA-based pesticide development and also helps revealing the insight of the plant-downy mildew interactions. |
Exploitation Route | Controlling downy mildew diseases are imperative for a sustainable agriculture. The target genes we identified can easily be used by companies to develop into RNA-based pathogen control. We have signed a nondisclosure Agreement with one of these companies and want to take it further. |
Sectors | Agriculture Food and Drink Chemicals Manufacturing including Industrial Biotechology |
URL | https://www.worcester.ac.uk/about/academic-schools/school-of-science-and-the-environment/science-and-the-environment-research/molecular-plant-and-microbial-biosciences-research-unit-mpmb-ru.aspx |
Description | EDGE FGT: RNAi-based tools to unlock functional genomics of obligate oomycete plant pathogens |
Amount | $400,000 (USD) |
Funding ID | 100000001 , 100015388 |
Organisation | National Science Foundation (NSF) |
Sector | Public |
Country | United States |
Start | 11/2023 |
End | 11/2024 |
Description | Bioinformatics |
Organisation | University of Exeter |
Department | College of Life and Environmental Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Provided biological samples for analysis |
Collaborator Contribution | Carried out bioinformatic analysis |
Impact | A formal collaboration started last year and RNA sequencing data has been produced by the collaborator for the project. This will be deposited to relevant databases in line with the data management part of the project in due time |
Start Year | 2022 |
Description | Producing small RNA in mini cell |
Organisation | Virginia Tech |
Country | United States |
Sector | Academic/University |
PI Contribution | We shared information on our sRNA project and explained our need |
Collaborator Contribution | They provided plasmid vectors, E.coli and methods to produce small RNA in E.coli. We applied for two grants together |
Impact | We applied for two grants together and decisions are pending for them |
Start Year | 2022 |
Description | siRNA mediated reverse genetics |
Organisation | Virginia Tech |
Country | United States |
Sector | Academic/University |
PI Contribution | We initiated whining review papers |
Collaborator Contribution | He brought his expertise to the review paper we wrote |
Impact | PMID: 36670035, PMID: 36061762 |
Start Year | 2022 |
Description | Application of small-RNA technologies in fundamental and translational research for plant protection: advantages, limitations and prospects |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | This was a keynote talk at an international conference held online. |
Year(s) Of Engagement Activity | 2021 |
URL | https://agbiol.org/files/46/editor/files/AGBIOL_2021_ABSTRACT_BOOK.pdf |
Description | Control of downy mildew pathogens using spray induced gene silencing. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | This talk was printed at the 2nd Molecular Plant Protection Congress, May 15-18, Bursa, Turkey. |
Year(s) Of Engagement Activity | 2023 |
Description | Controlling downy mildews using microbial products and ds RNA. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | This talk was presented at BSPP Plant Pathology 2023, 6-8 September, Birmingham, UK. |
Year(s) Of Engagement Activity | 2023 |
Description | Fundamental and translational research on downy mildews: Reverse genetics, pathogenomics and biologics |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Study participants or study members |
Results and Impact | This was in 21st Annual Meeting of the Oomycete Molecular Genetics Network. August, Brno, Czech Republic. Intended purpose was to give a kind of progress report on the projects to the learned society. |
Year(s) Of Engagement Activity | 2022 |
URL | http://www.phytophthora.org/coajdfadlf/uploads/2022/03/omgn2022.jpg |
Description | Fundamental and translational research on plant-downy mildew interactions. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | This was a talk at the Warwick University Departmental Seminar Series |
Year(s) Of Engagement Activity | 2023 |
Description | Pathogenomic-assisted plant breeding for disease resistance. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Study participants or study members |
Results and Impact | This was an online talk delivered to 1st International Congress on Biotech Studies. Intention was to publicise our work on Tony mildew pathogens and how it can be used for industry. |
Year(s) Of Engagement Activity | 2022 |
URL | https://icbios.org |
Description | Towards RNA-based downy mildew control in pea plants. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | This talk was presented at the UKLRC 2024 Annual Conference & PCGIN Annual Stakeholder meeting Programme, 14-15 Feb, Reading University. |
Year(s) Of Engagement Activity | 2024 |
URL | https://uklrc.org/uklrc2024/ |
Description | Use of Small Interfering RNAs As a Reverse Genetics Tools in Downy Mildew Pathogen |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | This talk was presented at the PAG Australia, International Plant and Animal Genome Conference. |
Year(s) Of Engagement Activity | 2023 |