Mechanisms of siRNA mediated broad-spectrum resistance to eukaryotic pathogens
Lead Research Organisation:
University of East Anglia
Department Name: Sainsbury Laboratory
Abstract
The sustainability of agriculture is threatened by pathogens, which cause substantial damage to crop yield and food safety. Although plants have evolved a myriad of immunity mechanisms to defend themselves, successful pathogens can overcome this surveillance system and cause disease. The dynamic interactions between virulence factors of pathogens and the innate immunity of a host determine whether disease will occur. To protect crops from pathogens, it is essential to identify new defense mechanisms and understand the molecular basis of their functions in order to design innovative approaches to elevate disease resistance.
Recent research discovered a small RNA (sRNA)-based defense mechanism in plants. Small RNAs are short RNA molecules (usually 20-24 nucleotide in length) that guide the inhibition of target gene expression based on sequence complementarity. Gene silencing or RNA interference (RNAi) triggered by sRNAs is a fundamental and universal regulatory mechanism in eukaryotes that impacts a wide range of biological processes. During host-pathogen interactions, sRNAs produced from one organism have been observed to affect gene expression in the opposing organism. Although this "trans-species" RNAi is an exciting concept that represents a new perspective in host-pathogen arms race, many challenges remain. A fundamental challenge is to identify which sRNAs in plant execute target genes silencing in invading pathogens. Furthermore, direct experimental evidence demonstrating gene silencing guided by plant sRNAs in invading pathogens is lacking. These major gaps of knowledge need to be filled before sRNAs can be effectively deployed in disease control.
In this project, we aim to investigate the mechanism underlying pathogen gene silencing by plant sRNAs during natural infection. This project builds on our recent discoveries suggesting that a specific family of plant sRNAs confer resistance to a filamentous eukaryotic pathogen. These sRNAs are produced from a unique biogenesis pathway that leads to the generation of a diverse sRNA pool, which has the potential to silence target genes in a broad range of eukaryotic pathogens. As such, we identified a designated family of "antimicrobial" sRNAs that may confer broad-spectrum resistance.
We will use a combination of genetics, molecular biology, biochemistry, synthetic biology, and plant pathology approaches and the model Arabidopsis thaliana-Phytophthora capsici pathosystem to investigate the function of this specific family of sRNAs in plant immunity. Trans-species gene silencing will be monitored by detection of plant sRNAs in the protein complex that guides gene silencing in the invading pathogen. We will test the hypothesis that this novel defense mechanism confers broad-spectrum resistance by examining plant mutants defective in sRNA production for susceptibility to additional eukaryotic pathogens. We will further explore how sRNA-spawning sequences could be edited to produce bespoke sRNAs that can increase pathogen gene silencing and elevate resistance.
The outcome of this project will be to provide novel insight into fundamental principles of plant immunity and offer new opportunities for the development of sustainable disease control strategies with far-reaching implications to a broad range of pathosystems.
Recent research discovered a small RNA (sRNA)-based defense mechanism in plants. Small RNAs are short RNA molecules (usually 20-24 nucleotide in length) that guide the inhibition of target gene expression based on sequence complementarity. Gene silencing or RNA interference (RNAi) triggered by sRNAs is a fundamental and universal regulatory mechanism in eukaryotes that impacts a wide range of biological processes. During host-pathogen interactions, sRNAs produced from one organism have been observed to affect gene expression in the opposing organism. Although this "trans-species" RNAi is an exciting concept that represents a new perspective in host-pathogen arms race, many challenges remain. A fundamental challenge is to identify which sRNAs in plant execute target genes silencing in invading pathogens. Furthermore, direct experimental evidence demonstrating gene silencing guided by plant sRNAs in invading pathogens is lacking. These major gaps of knowledge need to be filled before sRNAs can be effectively deployed in disease control.
In this project, we aim to investigate the mechanism underlying pathogen gene silencing by plant sRNAs during natural infection. This project builds on our recent discoveries suggesting that a specific family of plant sRNAs confer resistance to a filamentous eukaryotic pathogen. These sRNAs are produced from a unique biogenesis pathway that leads to the generation of a diverse sRNA pool, which has the potential to silence target genes in a broad range of eukaryotic pathogens. As such, we identified a designated family of "antimicrobial" sRNAs that may confer broad-spectrum resistance.
We will use a combination of genetics, molecular biology, biochemistry, synthetic biology, and plant pathology approaches and the model Arabidopsis thaliana-Phytophthora capsici pathosystem to investigate the function of this specific family of sRNAs in plant immunity. Trans-species gene silencing will be monitored by detection of plant sRNAs in the protein complex that guides gene silencing in the invading pathogen. We will test the hypothesis that this novel defense mechanism confers broad-spectrum resistance by examining plant mutants defective in sRNA production for susceptibility to additional eukaryotic pathogens. We will further explore how sRNA-spawning sequences could be edited to produce bespoke sRNAs that can increase pathogen gene silencing and elevate resistance.
The outcome of this project will be to provide novel insight into fundamental principles of plant immunity and offer new opportunities for the development of sustainable disease control strategies with far-reaching implications to a broad range of pathosystems.
Technical Summary
This project will investigate the burgeoning area of trans-species gene silencing during plant-pathogen interactions. We will characterize host-induced gene silencing (HIGS) mediated by secondary small interfering RNA (siRNAs) as a novel defense mechanism and determine its role in conferring broad-spectrum resistance to eukaryotic pathogens.
Plants produce a diverse pool of secondary siRNAs, which requires long double-stranded RNA precursors synthesized from specific endogenous transcripts. These precursors are then diced into an array of siRNAs. In eudicots, a main source of secondary siRNAs is a subset of PPR (pentatricopeptide repeat protein) genes. We recently discovered that PPR-derived siRNAs (PPR-siRNAs) of Arabidopsis thaliana confer resistance to the oomycete pathogen Phytophthora capsici, possibly through HIGS.
We will generate A. thaliana mutants using CRISPR/Cas9-based chromosomal deletion and target mimic-based sRNA inactivation to determine the role of PPR-siRNAs in plant defense. Trans-species gene silencing will be monitored by detecting PPR-siRNAs in the AGONAUTE complex of P. capsici. PPR-siRNAs include diverse sequences that potentially target genes in a broad range of pathogens. We will identify potential targets of PPR-siRNAs in two fungal pathogens and interrogate A. thaliana mutants defective in secondary siRNA and PPR-siRNA production for their contribution to broad-spectrum resistance. Finally, transgenic A. thaliana expressing edited PPR sequences that generate bespoke siRNAs targeting multiple sites within one gene or multiple genes in P. capsici will be tested for increased efficiency of target gene silencing and disease resistance.
This project will provide important new insight into the biology of plant immunity and pave the path for implementing siRNA-mediated defense into the on-going efforts of developing durable disease resistance in economically important crops.
Plants produce a diverse pool of secondary siRNAs, which requires long double-stranded RNA precursors synthesized from specific endogenous transcripts. These precursors are then diced into an array of siRNAs. In eudicots, a main source of secondary siRNAs is a subset of PPR (pentatricopeptide repeat protein) genes. We recently discovered that PPR-derived siRNAs (PPR-siRNAs) of Arabidopsis thaliana confer resistance to the oomycete pathogen Phytophthora capsici, possibly through HIGS.
We will generate A. thaliana mutants using CRISPR/Cas9-based chromosomal deletion and target mimic-based sRNA inactivation to determine the role of PPR-siRNAs in plant defense. Trans-species gene silencing will be monitored by detecting PPR-siRNAs in the AGONAUTE complex of P. capsici. PPR-siRNAs include diverse sequences that potentially target genes in a broad range of pathogens. We will identify potential targets of PPR-siRNAs in two fungal pathogens and interrogate A. thaliana mutants defective in secondary siRNA and PPR-siRNA production for their contribution to broad-spectrum resistance. Finally, transgenic A. thaliana expressing edited PPR sequences that generate bespoke siRNAs targeting multiple sites within one gene or multiple genes in P. capsici will be tested for increased efficiency of target gene silencing and disease resistance.
This project will provide important new insight into the biology of plant immunity and pave the path for implementing siRNA-mediated defense into the on-going efforts of developing durable disease resistance in economically important crops.
Publications
Gui X
(2022)
Phytophthora effector PSR1 hijacks the host pre-mRNA splicing machinery to modulate small RNA biogenesis and plant immunity.
in The Plant cell
Description | participated in training MSc students in the "Global Plant Health" program |
Geographic Reach | Multiple continents/international |
Policy Influence Type | Influenced training of practitioners or researchers |
Impact | We have five students from African countries who are trained through our MSc program in 2022-2023 academic year. This training will place them onto great positions for a career in academia, industry or policy making. |
Description | With Najing Agricultural University on Phytophthora genetic manipulation |
Organisation | Nanjing Agricultural University |
Country | China |
Sector | Academic/University |
PI Contribution | This is our project. We provide research concept, goal, materials and biological questions. |
Collaborator Contribution | Our collaborators are expert on Phytophthora genetics. They help us generate Phytophthora mutants. |
Impact | Not multiciplinary. Not outcome yet as the collaboration just started. |
Start Year | 2023 |
Description | BSPP 2022 (Newcastle), September 1, 2022 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | I participated the BSPP (British Society of Plan Pathology) presidential symposium in Newcastle and gave a talk on our research. Funding from BBSRC was acknowledged. |
Year(s) Of Engagement Activity | 2022 |
Description | Invited keynote speaker for Barcelona RNA club (April 21, 2022) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | I was invited to attend the annual symposium of the Barcelona RNA club (BaRNA) and gave a keynote talk. Funding from BBSRC was acknowledged. |
Year(s) Of Engagement Activity | 2022 |
Description | Invited keynote speaker for the COST Action symposium in Montpellier, October 20, 2022 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | I was invited to partcipitate a research symposium organized by INRA, France on molecular plant-microbe interactions. The audience included graduate students, postdocs, research fellows and group leaders. Funding from BBSRC was acknowledged. |
Year(s) Of Engagement Activity | 2022 |
Description | Invited talk (University of Edinburgh) April 25, 2022 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | I was invited to visit the University of Edinburgh and gave a research seminar. Funding from BBSRC was acknowledged. |
Year(s) Of Engagement Activity | 2022 |
Description | Invited talk (University of Oxford), June 9, 2022 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | I was invited to visit Oxford and gave a research seminar to the department of plant sciences. Funding from BBSRC was acknowledged. |
Year(s) Of Engagement Activity | 2022 |
Description | Invited talk (University of York), November 29, 2022 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | I was invited to give a research seminar on our research related to this funded project. Funding from BBSRC was acknowledged. |
Year(s) Of Engagement Activity | 2022 |
Description | Invited talk (University of Zurich), November 11, 2022 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | I gave an invited research seminar at the University of Zurich. In the audience were undergraduate/graduate students, postdoc fellows and group leaders. Funding from BBSRC was acknowledged. |
Year(s) Of Engagement Activity | 2022 |
Description | Online seminar for aBIOTECH, November 18, 2022 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | I was invited as a keynote speaker for an online symposium on plant biotechnology organized by the journal aBIOTECH. This symposium attracted a broad audience with >3,000 attendees globally. Funding from BBSRC was acknowledged. |
Year(s) Of Engagement Activity | 2022 |
Description | Online seminar for the University of Washington (Seattle) April 11, 2022 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | I was invited to give an online research seminar to the University of Washington. Funding from BBSRC was acknowledged. |
Year(s) Of Engagement Activity | 2022 |
Description | Participation in the Oomycete molecular genetic network symposium (Brno) August 23, 2022 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | I attended and gave a talk at the oomycete molecular genetics network symposium held in Brno, Cezch Republic. My talk was closely related to the projects funded by BBSRC. Funding from BBSRC was acknowledged. |
Year(s) Of Engagement Activity | 2022 |
Description | Public outreach - Plants of the future (May 21, 2022) |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | This was an open day at the Norwich Research Park with scientific exhibits, tours and family-friendly activities. I helped designed and participated in two activities (observing plant pathogens through microscope and DNA extraction), which attracted many visitors. |
Year(s) Of Engagement Activity | 2022 |